Polyamine compounds and compositions for use in conjection with cancer therapy

ABSTRACT

The invention provides novel polyamine compounds and pharmaceutical compositions for administration in conjunction with cancer chemotherapy or radiation therapy. The compounds are administered locally to provide protection against the adverse side-effects of chemotherapy or radiation therapy, such as alopecia, mucositis and dermatitis. Pharmaceutical preparations comprising one or more chemoprotective polyamines formulated for topical or local delivery to epithelial or mucosal cells are disclosed. Methods of administering the pharmaceutical preparations are also disclosed.

This application claims benefit of U.S. Provisional Application No.60/355,356, filed Feb. 7, 2002, the entirety of which is incorporated byreference herein.

Pursuant to 35 U.S.C. §202 (c), it is acknowledged that the UnitedStates Government has certain rights in the invention described herein,which was made in part with funds from the National Institutes ofHealth, Grant No. CA22484.

FIELD OF TIRE INVENTION

The present invention relates to the field of cancer therapy. Inparticular, the invention provides novel polyamine compounds andpharmaceutical compositions for reducing or preventing toxic sideeffects of radiotherapy and cancer chemotherapeutic agents.

BACKGROUND OF THE INVENTION

Various patents and other publications are referenced in thisapplication in order to more fullly describe the state of the art towhich this invention pertains. The disclosure of each of thesepublications is incorporated by reference herein, in its entirety.

It is well known that the use of chemotherapy and radiotherapy to treatcancer patients is associated with severe side effects due to thetoxicity of such treatments to epithelial cell populations, includingstem cells within the hair follicle, skin epidermis and gastrointestinalmucosa

Currently, there are no treatments to prevent cancer therapy sideeffects. Effective treatments would likely include molecules that i)inhibit or slow growth of the at-risk cells, ii) modify the cellular DNAof the at-risk cells to make it less easily damaged, and iii) providesome means with which to scavenge electrophilic drug metabolites oroxygen radicals formed during irradiation

Polyamines have been proposed as growth regulators. DENSPM, a syntheticanalog of spermine, has been shown to decrease cell growth (Kramer etal., Cancer Res. 57:5521-5527, 1997), and has been studied in an earlystage clinical trial as an antineoplastic drug (Creaven, P. et al.,Invest. New Drugs 15:227-234, 1997; Streiff, R and Bender, J., Invest.New Drugs 19:29-39, 2001). The clinical trials, however, were abortedbecause of the serious side effects in multiple organ sites that wereassociated with the systemic use of this polyamine analog. These resultsteach that molecules used to decrease division of healthy stem cellsthat are at risk from cancer therapy would need to create a transientcell cycle block and would need to be applied topically to achieve localdelivery to epithelial cells, with little or no systemic delivery, or ifany, low enough to preclude protection of systemic cancer cells orinduction of systemic side effects.

Naturally occurring polyamines, such as spermine, have been shown tobind to nucleic acids and to induce structural changes in helical DNA(Basu, H. and Marton, L., Biochem. J. 244:243-246, 1987; Feuerstein, B.et al., Nuc. Acids Res. 17:68836892, 1989). This binding has beensuggested to occur through interaction of the positively charged aminegroups in the polyamine backbone and negatively charged sites on the DNAbackbone. Because of the nature by which electrophilic chemotherapydrugs or oxygen radicals generated by radiotherapy attack helical B-DNAwithin cells, the ability of polyamines to bind DNA and disrupt normalB-DNA structure could be helpful in protecting DNA within cells to whicha polyamine was delivered.

An additional strategy for protecting cells againstelectrophiles/radicals has been to augment levels of the naturallyoccurring cellular nucleophile, glutathione (GSH). Both animal and cellculture studies have shown that there is a direct relationship betweenthe intracellular concentration of GSH and the amount of exogenouslyadministered alkylating molecule that is needed to achieve cell kill(Ho, D. and Fahl, W., J. Biol. Chem. 259:11231-11235, 1984;Ellouk-Achard, S. et al., Arch. Toxicol. Suppl. 17:209-214, 1995).Efforts to exogenously administer GSH to cells as a protectant havefailed because mammalian cells are generally unable to take up thisnucleophile. There have been efforts to modify the GSH molecule toenable cellular uptake, but these have not found clinical use.

Amifostine (WR-2721), a small molecule amine containing a thiophosphategroup that is presumably converted to a thiol in cells, has been usedsystemically as a radio- and chemoprotectant with mixed results. Thoughit may provide free —SH groups within cells, it is not known to containactivity as either a growth regulator or as a modifier of DNA structure.

Edwards et al. (U.S. Pat. Nos. 5,217,964 and 5,434,145) described thesynthesis of short, spermidine- or spermine-like polyamine moleculesthat were modified to contain an alkyl-thiophosphate or alkyl-thiolgroup. In U.S. Pat. No. 5,217,964, the attached thiophosphate group(i.e., —SPO₃H₂) would require enzymatic activation by cellularphosphatases to form the nucleophilic —SH group. The alkyl-thiophosphategroup(s) is bound to the polyamine molecule through a terminal benzylring and/or through one or more of the amines in the polyamine backbone.Polyamines containing aromatic rings have been described in the art tobe structural inhibitors of the membrane polyamine transporter inmammalian cells and have been shown, themselves, not to be transportedinto cells. In U.S. Pat. No. 5,434,145, Edwards showed bonding ofalkyl-thiophosphate or alkyl-thiol groups to one or more of the backboneamines that are present in the short polyamine molecules. By modifyingthe secondary amines in the polyamine backbone with alkyl-thiophosphategroups, the amines were converted to tertiary amines, and this markedlyaltered the basicity of the individual modified amine, as well as theoverall polyamine molecule. The attenuated basicity of the individualamine groups was accompanied by an alteration in 3-dimensional structureat these sites. With added alkyl functionality on the amine nitrogenatoms, steric bulkiness increased, so the ability or freedom of themolecule to rotate and twist at these sites was markedly reduced. Thealtered basicity and steric constraints in these short spermine-likepolyamines was surmised to perturb DNA binding by the polyamine ascompared to their natural polyamine counterparts. Consistent with this(DNA binding is a biological activity of natural polyamines), Edwardsprovided no information regarding biological activity for any of thestructures proposed in U.S. Pat. Nos. 5,217,964 or 5,434,145.

There is a need in the art, then, to create polyamine-based moleculesthat are optimized to achieve: i) local and transient growth regulation,ii) disruption of normal helical DNA structure upon binding, and iii)delivery and display of nucleophilic or other functional moieties withincells to enable scavenging of reactive electrophiles and radicals. Therewould be great advantage in developing polyamine derivatives that couldbe used topically to prevent or diminish the toxic side effects ofcancer chemotherapy and radiotherapy.

SUMMARY OF THE INVENTION

The present invention provides novel polyamine compounds andpharmaceutical compositions for reducing or preventing toxic sideeffects of radiotherapy and cancer chemotherapeutic agents. Thepolyamine compounds of the invention are referred to herein as“chemoprotective polyamines.”

One aspect of the invention fears a compound of Formula I:

-   -   wherein:    -   each Z is independently A or R¹;    -   each A is independently:    -   J is a single bond or —CH(Y)—;    -   X is D or —R²-D;    -   Y is H, alkyl, or R³-D;    -   D is —OH, —SH, —SR⁴, or —NR⁴R⁵;    -   each R¹ is independently C₃alkylene;    -   each R², R³, R⁶, and R⁷ is independently C₁₋₆ alkylene;    -   R⁴ is H or lower alkyl;    -   R⁵ is H, lower alkyl, or —R⁶-D;    -   Q is H, lower alkyl or —R⁷—SR⁴;    -   k is an integer from 2 to about 16;    -   or a stereoisomer, prodrug, pharmaceutically acceptable salt, or        mono or polyprotonated acid salt thereof.

In one embodiment, each A is independently:

In this embodiment, Y may be H or R³-D. X may be D or R²-D. In thisembodiment, k is an integer from 2 to about 16. In specific embodiments,k is 2, 3, 4, 5, 6, 7 or 8. In other specific embodiments, k is 2-8,each R¹ is butylene, X is D, D is —NR⁴R⁵, R⁴ is H, and R⁵ is ethyl, andQ is ethyl. In yet other specific embodiments, k is 2, 4, 6 or 8, eachR¹ is butylene, X is D, D is —SH, and Q is ethyl. Yet another specificembodiment comprises a compound wherein k is 4, each R¹ is butylene, Xis D, D is —NR⁴R⁵, R⁴ is H, R⁵ is methyl, and Q is ethyl. In otherembodiments, Q is H or lower alkyl. Exemplary compounds having thesefeatures are shown in FIG. 1A through FIG. 1C.

In another embodiment, each A is independently:

In this embodiment, Y may be H or R³-D. X may be D or R²-D. In thisembodiment, k is an integer from 2 to about 16. In specific embodiments,k is 2, 3, 4, 5, 6, 7 or 8. Q may be H or lower alkyl. J is a singlebond; in specific embodiments, J is —CH(Y)—. Exemplary compounds havingthe aforementioned features are shown in FIG. 1D and FIG. 1E.

Another aspect of the invention features a pharmaceutical preparationfor reducing or preventing hair loss, dermatitis, mucositis orgastrointestinal distress caused by treatment with a chemotherapeuticagent or radiation therapy, which comprises at least one compound ofFormula I as described above, and a topical delivery vehicle for locallydelivering the compound to dermal or mucosal cells of skin, scalp,mouth, nasoesophageal, gastrointestinal or urogenital system. In certainembodiments, the pharmaceutical preparation further comprises at leastone other agent that reduces or prevents hair loss, dermatitis,mucositis or gastrointestinal distress caused by treatment with achemotherapeutic agent or radiation therapy, for instance, ananti-proliferative agent, a chemoprotective inducing agent or a freeradical scavenger.

The topical delivery vehicle comprises one or more of liposomes, lipiddroplet emulsions, oils, aqueous emulsions of polyoxyethylene ethers,aqueous alcohol mixtures, aqueous ethanol mixtures containing propyleneglycol, aqueous ethanol mixtures containing phosphatidyl choline,lysophosphatidyl choline and triglycerides, xanthan gum in aqueousbuffer, hydroxypropymethylcellulose in aqueous buffer or aqueous alcoholmixtures, diethylene glycol monoethyl ether in aqueous buffer, andbiodegradable microparticles.

In a specific embodiment, the pharmaceutical preparation is formulatedfor topical delivery to skin or hair follicles, and the delivery vehiclecomprises an aqueous alcohol mixture and, optionally, propylene glycol.Preparations of this type may be formulated as creams, lotions,ointments or gels. In another specific embodiment, the pharmaceuticalpreparation is formulated for topical delivery to the oral cavity ornaso-esophageal passages. In this embodiment the delivery vehiclepreferably comprises a mucoadhesive substance. It may be formulated asan aerosol, oral rinse, ointment or gel. In yet another specificembodiment, the pharmaceutical preparation is formulated for vaginal orrectal delivery and comprises a mucoadhesive substance. Thesepreparations may be formulated as creams, ointments, lotions, gels,foams or suppositories. In still another specific embodiment, thepharmaceutical preparation is formulated for topical delivery to thegastrointestinal tract and the delivery vehicle comprises one or more ofnonionic liposomes and mucoadhesive substances. Preferably, thepreparation is formulated as a liquid for coating the surface of thegastrointestinal tract.

According to another aspect of the invention, methods are provided forreducing or preventing hair loss dermatitis, mucositis orgastrointestinal distress in a patient undergoing treatment with achemotherapeutic agent or radiation therapy. The methods compriseadministering to the patient a pharmaceutical preparation as describedabove, in an amount and for a time sufficient to reduce or prevent thehair loss, dermatitis, mucositis or gastrointestinal distress. In oneembodiment, the pharmaceutical preparation is administered beginning atleast one day, and preferably up to five or more days, prior tochemotherapy or radiation therapy. In another embodiment, thepharmaceutical preparation is administered after initiation ofchemotherapy or radiation therapy. Preferably, the pharmaceuticalpreparation is administered throughout a course of chemotherapy orradiation therapy and, in certain instances continues after terminationof a course of chemotherapy or radiation therapy.

The aforementioned methods may further comprise administering to thepatient at least one other agent that reduces or prevents hair loss,dermatitis, mucositis or gastrointestinal distress caused by treatmentwith a chemotherapeutic agent or radiation therapy. These other agentsmay include anti-proliferative agents, chemoprotective inducing agentsor free radical scavengers, for instance.

The present invention also provides a method of treating cancer thatincreases a patient's tolerance to high doses of a chemotherapeuticagent or radiation therapy. The method comprises (a) administering thehigh dose of the chemotherapeutic agent or radiation therapy to thepatient; and (b) administering one or more of the above-describedpharmaceutical preparations for reducing or preventing one or more ofchemotherapy- or radiation therapy-induced hair loss, dermatitis,mucositis or gastrointestinal distress, in an amount and for a time toreduce or prevent the one or more of the chemotherapy- or radiationtherapy-induced hair loss, dermatitis, mucositis or gastrointestinaldistress, thereby increasing the patient's tolerance to the high dose ofthe chemotherapeutic agent or radiation therapy.

Other features and advantages of the present invention will beunderstood by reference to the drawings, detailed description andexamples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1E illustrate the structures of certain of thechemoprotective polyamine molecules whose synthetic pathways areillustrated in the reaction schemes. FIG. 1A shows compounds PrC 110,111, 112 and 113, olefinic core displaying —NH—CH₂—CH₃ functional group;FIG. 1B shows compounds PrC 114,115,116, 117 and 118, olefinic coredisplaying SH or H functional group; FIG. 1C shows compounds PrC 119,120, 121, 122 and 123, olefinic core displaying —NHCH₃, —N(CH₃)₂ or SHfunctional group; FIG. 1D shows compounds PrC 210, 211, 212, 213 and214, aliphatic core displaying —OH, —SH, —SCH₃ or —NHCH₂CH₃ functionalgroup; FIG. 1E shows compounds PrC 215, 216, 217 and 218, aliphatic coredisplaying —OH, —SH, —SCH₃ or SCH₂CH₂N(CH₃)₂ functional group.

FIG. 2 illustrates the relationship between the number of aliphaticcarbon atoms in each chemoprotective polyamine side chain (‘arm’) andthe respective IC₅₀ dose for inhibition of human fibroblast growth.

FIGS. 3A and 3B illustrate the level of induced p21 protein seen indiploid human fibroblasts after a 30 hr exposure to each of theindicated chemoprotective polyamines. FIG. 3B shows that the induced p21level is greater after a 30 hr exposure compared to a 50 hr exposure todrug. In these experiments, the 23SK human skin cells were exposed for30 hr to an “IC_(so)” dose of each of the indicated chemoprotectivepolyamines and then lysed. Cell extracts were then prepared in order tomeasure p21 levels by western analysis (FIG. 3A).

FIG. 4 illustrates the relationship between the number of aliphaticcarbon atoms in each chemoprotective polyamine ‘arm’ and the respectiveinduced p21 level in diploid human fibroblasts after a 30 hr exposure.The arrow points to the value for PrC-110, which also showed excellentefficacy in the in vivo alopecia test.

FIGS. 5A-5D are cell histograms showing the results from flow cytometryanalysis of chemoprotective polyamine-treated 23SK skin cells. FIG. 5Ashows results from untreated, exponentially growing 23SK cells. FIG. 5Bshows, as a control treatment, results from incubation of cells inserum-free medium. FIG. 5C shows results from cells treated with PrC-117for 72 hr. FIG. 5D shows results from cells treated with PrC-117 for 72hr, then switched for 48 hr to um devoid of the PrC-117 molecule.

FIGS. 6A-6E illustrate the efficacy of topically-applied chemoprotectivepolyamines in protecting against chemotherapy-induced alopecia (hairloss) in a rodent model.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides compounds for use in pharmaceuticalpreparations and methods for protecting non-cancerous, rapidly dividingcells in a patient's body from the toxic effects of chemotherapeuticagents or radiotherapy administered to the patient. In particular, thecompositions and methods of the invention are designed for protectingepithelial cells. Most particularly, the targets are epithelial cellslining hair follicles and epithelial and/or mucosal cells of the skin,mouth, gastrointestinal (GI) and urogenital tract In one embodiment, thecompositions are used to reduce or prevent alopecia during cancertherapy, by topically applying the composition to the scalp. Anotherembodiment comprises reduction or prevention of gastrointestinaldistress due to cancer therapy by administering the compositions orally.Another embodiment involves reducing or preventing mucositis fromchemotherapy or radiotherapy by administering the compositions topicallyto the appropriate region of the body. In yet another embodiment, thecompositions are used to prevent radiation-induced dermatitis, skinrash, and ulceration at the site of irradiation by applying them to theskin.

The chemotherapeutic agents from which protection of normal cells isdesired may be one or a combination of agents used for such purpose,such as alkylating agents, antimetabolite inhibitors of DNA synthesis,antitumor antibiotics, mitotic spindle poisons, vinca alkaloids, andtopisomerase inhibitors. Specific chemotherapeutic agents include, butare not limited to, altretamine, asparaginase, bleomycin, busulin,carboplatin, cispiatin, carmustine, chlorambucil, cladribine,cyclophosphamide(cytoxan), cytarabine, dacarbazine, dactinomycin,daunorubicin, doxorubicin, etoposide, floxuridine, fludarabinephosphate, fluorouracil, hydroxyurea, idarubicin, ifosfamide, lomustine,mechlorethamine, nitrogen mustard, melphalan, mercaptopurine,methotrexate, mitomycin, mitoxantrone, paclitaxel pentostatin,pliamycin, procarbazine, streptozocin, teniposide, thioguanine,thiotepa, vinblastine and vincristine. The radiation therapy consists ofall useful types of radiation used in cancer treatment, includingx-rays, gamma-rays, electron beams, photons, alpha-particles andneutrons.

Commonly-owned, co-pending U.S. application Ser. No. 10/214,917 andInternational Application No. PCT/US02/25216, each filed Aug. 7, 2002,describe that several types of known polyamines and polyamine analogs,referred to therein as “polyamine effector” compounds, can beefficiently delivered to the aforementioned target cell populations,where they are capable of protecting those cells from the harmful sideeffects of chemotherapy or radiotherapy. The present invention providesnovel polyamine compounds specifically designed for improved efficacy inprotecting normal cells from the detrimental effect of cancerchemotherapy or radiation therapy. These molecules are referred toherein as “chemoprotective polyamines.”

Certain definitions that will assist in the understanding of the presentinvention are set forth below, while others are provided throughout thespecification. With respect to the compounds of the invention, it shouldbe noted that if any variable occurs more than one time in anyconstituent or in any formula, its definition in each occurrence isindependent of its definition at every other occurrence. Thus, forexample, if a compound of the present invention is shown to incorporate,for example, one or more of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, —OH, —SH, —SR⁴,or —NR⁴R⁵, then the R¹, R², R³, R⁴, R⁵, R⁶, R⁷, —OH, —SH, —SR⁴, or—NR⁴R⁵ at each occurrence is selected independently. Combinations of R¹,R², R³, R⁴, R⁵, R⁶, R⁷, —OH, —SH, —SR⁴, or —NR⁴R⁵ are permissible onlyif such combinations result in stable compounds.

Polyamines are small aliphatic amines found in all living cells. Bynature, polyamines within cells are polycationic (i.e., capable ofsustaining or neutralizing one or more equivalents of acid). They atebiosynthesized from amino acids, such as arginine and omithine. Examplesof common polyamines found in plant and animal cells are: putrescine(NH₂(CH₂)₃NH₂), formed by the decarboxylation of ornithine or arginine;spermidine (NH₂(CH₂)₄NH(CH₂)₄NH₂); and spermine(NH₂(CH₂)₃NH(CH₂)₄NH(CH)₃NH₂); the latter two being formed by subsequentaddition of an aminopropyl moiety to putrescine and spermidine,respectively. Because such polyamines are found in nature, they may bereferred to as “naturally-occurring” polyamines. However, they may beprepared by a variety of synthetic strategies, as would be known in thechemical arts.

The term “polyamine analogs” as used herein refers to polycationicmolecules that are similar, but not identical to polyamines found innature. Polyamine analogs may be branched or unbranched, or may haveother structural variations as compared to naturally-occurringpolyamines, while retaining the central features of polyamines (multipleamine groups, polycationic within cells). Polyamine analogs may befurther categorized into three groups: (1) simple polyamine analogs, (2)constrained or conformationally restricted polyamine analogs, and (3)linked or long-chain polyamine analogs.

A “simple polyamine analog” retains the flexibility conferred by thealiphatic carbon backbone, as well as the approximate carbon chainlength of naturally-occurring polyamines, but possess a modification orcontain one or more added functional groups (e.g., sulfhydryl, phenyl,alkyl) that confers a desired feature or advantage to the molecule.

By comparison, “conformationally restricted polyamine analogs”(sometimes referred to herein as “constrained polyamine analogs” aremodified in their carbon backbone to remove flexibility in the modifiedarea, such that two or more amino functionalities in the molecule arerestricted to a particular spatial location. Such modification often isaccomplished by introducing a cyclic or unsaturated moiety at one ormore locations in the carbon backbone, as described in greater detailherein.

“Linked or long-chain polyamine analogs” are polyamines that are longerthan naturally-occurring polyamines such as spermine. Increasing theoverall length of a polyamine may be accomplished, for example, bylinking together oligoamines or by adding oligoamine “units” (such asaminopropyl or aminobutyl groups) to a foundation molecule, such asspermine. Thus, while spermine has a 3-4-3 carbon backbone (4 carbonsbetween the two internal amino groups and 3 carbons between eachinternal amino group and the respective terminal amino groups), linkedor long-chain analogs might comprise an additional one, two, three, fouror more aminopropyl or aminobutyl groups, for example, on either or bothends of the molecule, and further may comprise terminal methyl or ethylgroups on either or both ends.

As used herein, the term “antiproliferative” refers to an agent thatslows or stops cell division. The antiproliferative agent may exert itseffect by inhibiting cell cycle progression at one or more stages. Suchan agent may be referred to herein as a “cell cycle progressioninhibitor.” The chemoprotective polyamines of the invention can act asantiproliferatives, specifically cell cycle progression inhibitors, byassociating with and modifying the conformation or structure of DNA.These agents are sometimes referred to herein as “DNA modifiers.”

The design of the chemoprotective polyamines of the present inventionemerges from the inventors' appreciation of the advantages associatedwith blending certain important chemical properties within a singlemultifunctional molecule, 1) molecular structure necessary for efficientbinding to DNA and, in some instances, modification of the conformationor structure of DNA; 2) nucleophilic reactivity, to trap electrophilicchemicals that can challenge the integrity of helical DNA; and/or 3)free radical-scavenging activity to reduce or eliminate free radicalsoften generated by irradiation or various chemotherapeutic agents (e.g.,certain reactive oxygen species).

In regard to structure, the ability of a polyamine to physically alignclosely, or “dock” with DNA should be maintained. Mimicking the generallinear nature of the known natural polyamines enables thechemoprotective polyamines of the invention to maintain DNA bindingability. Another important feature common to natural polyamines is thepresence of multiple secondary amine nitrogen atoms throughout thebackbone. These atoms are known to be protonated, and thus positivelycharged, at physiologic pH. Accordingly, maintaining secondary aminefunctionality throughout a chemoprotective polyamine further providessufficient active binding sites.

Nucleophilic and/or free radical-scavenging activity was designed intothe chemoprotective polyamines with the aim of maintaining all of theabove mentioned structural and binding features. In various exemplaryembodiments described herein, electron-rich groups, bearingsp3-hybridized nitrogen, sulfur or oxygen atoms, were positionedstrategically within the polyamine backbone so that overall linearityand secondary amine character would be preserved for efficient DNAbinding. The enhanced reactivity of allylic functional groups, comparedto their alkyl counterparts, was also considered in designing placementof functional groups in certain embodiments. In some embodiments,chemoprotective polyamines with an olefinic core have thenucleophiles/scavengers positioned on allylic positions specifically toenhance the reactivity of those functional groups. In these embodiments,the core segment bearing the functional group was restricted in size,consistent with natural polyamine features, and provides a suitableplatform from which the nucleophile or other functional group isdisplayed. This design feature allows one side, or face, of the3-dimensional polyamine structure to interact with DNA while the otherface, bearing the reactive functional group, is projected away from theDNA, sterically unencumbered, thus free to react with toxicelectrophilic chemicals or free radicals present in the cellular matrix.

The chemoprotective polyamines of the present invention are representedby the general structure of Formula I:

In Formula I, “Z” is either “A” or “R¹.” “A” represents a “core” segmentand the R¹ and Q groups typically represent alkylene (R¹) or alkyl (Q)chains of varying length (branched or unbranched), which, together withthe amine groups as shown, make up the linked oligoamine segments thatform the polyamines of the present invention.

As used herein, “alkylene” refers to a bivalent alkyl radical having thegeneral formula —(CH₂)_(n)—, where n is 1 to about 8. Non-limitingexamples include methylene, ethylene, trimethylene, butylene,pentamethylene, and hexamethylene. Alkylene groups may be branched orunbranched. Alkylene groups may also contain one or more double ortriple bonds within the backbone of the —(CH₂)_(n)— moiety, providedthat the resultant compound is stable. Non-limiting examples include—CH₂—C≡C—CH₂— and CH₂—CH═CH—CH₂—. Alkylene groups can be substituted orunsubstituted, provided that the resultant compound is stable and solong as the substituent does not substantially interfere with presentcompound's intended mode of action. In certain circumstances, alkyleneis preferably C₃₋₈ alkylene, while in other circumstances, even withinthe same molecule, alkylene is preferably C₁₋₆ alkylene.

As used herein, “alkyl” refers to a saturated straight or branchedhydrocarbon having from about 1 to about 20 carbon atoms (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), with from about 1 to about 8 carbon atoms, hereinreferred to as “lower alkyl”, being preferred. Alkyl groups include, butare not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl,3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl, octyl, decyl,dodecyl, octadecanyl, and eicosanyl.

The core segment (“A”) functions in two ways: (1) it presents a platformfor display of a protective functional group, namely a nucleophile or afree radical scavenger; and (2) it may be designed to introduce aconformational constraint to the polyamine (e.g., a double bond or acyclic structure). The linked oligoamine segments (sometimes referred toas “arms” or as “polyamine side chains”) function to enable the moleculeto “dock” with DNA, as do naturally occurring polyamines. In oneembodiment, a compound of the invention comprises one core and an “arm”of varying length on either side of the core. In another embodiment, thecore may have a single arm (i.e., the core group is at one end or theother of the polyamine molecule). In another embodiment, thechemoprotective polyamine comprises two or more cores (which may be thesame or different), which can be side-by-side or separated by anoligoamine segment of varying length

The core segment provides the molecule with conformational restraintand/or a protective functional group that is attached (“tethered”) tothe molecule in such a way as to be optimally available for interactionwith electrophilic groups, free radical groups and other reactivespecies present on or generated by chemotherapeutic agents or radiation.In the present invention, conformation restraint is typically introducedthrough the use of a double bond between two carbons. As would beappreciated by those of skill in the art, other means of introducingconformational restraint include triple bonds and ring structures, suchas three-, four-, five- and six-carbon or more substituted orunsubstituted rings (in the latter embodiments, with the proviso thatthe ring does not introduce bulk or steric hindrance that reduces theability of the functional group to access its targets).

The protective functional groups displayed on the core are designed toact as nucleophiles or as free radical scavengers/antioxidants, with theunderstanding that certain functional groups may carry out bothfunctions. Functional groups that typically act as nucleophiles, butthat may also act as antioxidants or free radical scavengers, include,but are not limited to, —OH, —NH₂, —NHR, NR₂, —SH and —SR (wherein R ismethyl or a lower alkyl which itself may be substituted with —OH, —NH₂,—NR, NR₂, —SH or —SR).

The total length or size of a chemoprotective polyamine of the inventionis generally described herein by the number of oligoamine segments(R¹—NH—) that make up the molecule. Typically the compounds comprise twoor more such segments, and may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or even more such segments. The overall upper limit tolength of the compounds is typically selected on the basis of practicalconsiderations such as cost and ease of synthesis, solubility and/orskin or mucosal permeability, as measured against efficacy of thecompound in exerting its protective effect within the cell. In specificembodiments, the chemoprotective polyamine comprises 2, 3, 4, 5, 6, 7 or8 oligoamine segments.

1. Synthesis of Chemoprotective Polyamines Comprising Nucleophilic Cores

The synthetic approaches illustrated below demonstrate versatilityregarding the choice of nucleophile incorporated into the core segments,the availability of both cis- and trans-isomers of the olefinic core andvariability in the amine side chain segment length as well as number ofsegments desired Several reaction schemes and tables are presentedthroughout the sections below, with both reaction intermediates andfinal products being assigned unique descriptive numbers. Specificdescriptions of the synthesis of the key molecules are set forth inExample 1.

1.1 Amine Side Chains

In exemplary embodiments of the invention, amine side chains weresynthesized using the reaction sequences in Scheme 1. Primary alkylamine 1 was converted to mesitylene sulfonamide 2, which was alkylatedto provide N-phthaloyl protected 3. It should be noted that the segmentlength can be adjusted from two carbons to six carbons in this sequenceof steps, and this invention is not limited to the four-carbon chainlength of molecule 3. Deprotection of the terminal nitrogen gave 4,which was readily converted to 5. The bis-sulfonamide 5 represents theshortest amine side chain with regard to number of segments. Molecule 5also was used for chain elongation by adding segments. The threereaction steps that convert 2 to 5 were repeated and therefore representan iterative process by which 5 was converted to 8, 8 elaborated to 11and 11 to 14. Each of the mesitylenesulfonyl protected amine side chains5, 8, 11, 14, and related chain-extended derivatives, are suitable forattachment to a core segment. In sum, Scheme I describes how a singlepolyamine side chain may be produced. This process can be repeated toadd additional polyamine side chain segments.

1.2 Synthesis of Olefinic Core and Side Chain Attachment

A general description of the olefinic core synthesis is illustrated inScheme 2. Dihydroxyacetone dimer 15 was converted to ketone 16.Olefination of 16 provided ester 17, which was carefully reduced to theallylic alcohol 18 while maintaining the integrity of the silyl groups.Mesylation gave allylic mesylate 19, which was coupled to an amine sidechain to provide 20, where A represents the mesitylenesulfonyl protectedamine side chain. Acid treatment of 20 gave diol 21, which wasmonobenzoylated to provide 22. It should be noted that the cis- andtrans-isomers of alcohol 22 can be separated by chromatography toprovide the individual purified isomers. Alcohol 22 was then transformedto the allylic bromide 23, which was coupled to a second protected amineside chain to produce 24. For the purpose of this invention it should benoted that in 24, protected amine side chains A and A′ can be identical,but can also vary in segment length as well as overall chain length.Hydrolysis of 24 gave mesitylenesulfonyl protected polyamine 25.Protected polyamine 25 can be deprotected (see polyamine 27 in Scheme3), or serve as a versatile intermediate that can be further elaboratedat the allylic alcohol position to insert alternative protectivefunctional groups.

1.3 Functional Groups on the Chemoprotective Polyamine Core

A method for introducing various protective functional groups onto acore segment is shown in Scheme 3. Alcohol 26 was converted to mesylate27, which was subsequently reacted with various species having suitablenucleophilic character, to provide, for example, 29, 31, 33 or 35.Subsequent deprotection produced, for example, the chemoprotectivepolyamines 30, 32, 34 and 36.

1.4 Functional Groups Displayed from an Aliphatic Core

A synthetic approach to chemoprotective polyamines bearing functionalgroups on an aliphatic core segment is shown in Scheme 4.

In some pharmacologic settings, there may be advantage in displaying aprotective functional group from a flexible aliphatic core as has beendone in molecules PrC-210, PrC-211, as well as the rest of the moleculesshown in FIGS. 1D and 1E. Using chemoprotective polyamines to delivernucleophiles/free radical scavengers to at-risk cells, while alsobinding DNA to enable DNA protection and growth regulation, requiresoptimization of each of the chemoprotective polyamine's structuralparameters, including segment length, overall length, functional group,and the platform from which the functional group is displayed. Forinstance, displaying an alkyl-nucleophile side chain from a flexiblecore may change the interaction between polyamine and DNA, and with it,change the growth regulation “phenotype” that would be linked with thedisplayed nucleophile “phenotype” on a particular chemoprotectivepolyamine. This combination of functions within a given molecule may beoptimized for each pharmacologic use of chemoprotective polyamines. Inthe reaction sequence of Scheme 4, dichloride 37 was converted to olefin38, which was subsequently transformed to alcohol 39. Alcohol 39 can bedeprotected to give 40, or converted to the mesylate intermediate 41.Mesylate 41 was then converted, with suitable nucleophiles, to 42 and44, which upon deprotection, produce chemoprotective polyamines 43 and45.

Other aliphatic polyamines may be prepared by hydrogenating olefinicpolyamines of the invention. This is accomplished by employinghydrogenation catalysts in the presence of hydrogen or molecules thatprovide hydrogen during the course of a reaction, such as for example,hydrazine, cyclohexadiene, or alpha-terpinene. Further, as oneordinarily skilled in the art would recognize, one or more of the doublebonds in any given olefinic polyamine may be selectively hydrogenated byselection of catalysts that preferably coordinate to one or more of the“D” moieties of the present compounds and transfer hydrogen selectivelyto the olefin adjacent to the “D” moiety. For a general overview, see J.March, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure,Fourth Edition, John Wiley and Sons, New York (1992), pp 771-780.

1.5 Polyamines Containing Two or More Cores

The synthesis of a chemoprotective polyamine with more than one coreunit is illustrated in Scheme 5. The core intermediate 23 (see Scheme 2)is reacted with sulfonamide 54 to give 55. Removal of the phthaloylgroup provides 56, which upon sufonylation gives bis-sulfonamide 57. Ifthe desired functional group on the core segments of the targetpolyamine is hydroxyl, 57 can be converted directly to silyl ether 60,where X═OH. Alternatively, the nucleophile can be modified by converting57 to 58, followed by functional group transformation to 59. Sulfonamide59 can likewise be converted to 60. Desilylation to 61, and subsequentbenzoylation, gives 62. Conversion to the bromide 63 provides a pivitolintermediate. The chain-elongation process can be terminated byattaching an amine side chain, thus providing a polyamine bearing twocore units. Alternatively, bromide 63 can be subjected to an iterativeprocess that involves repeating the steps shown at the start of Scheme5. This will install a third linker-core repeating unit in the polyaminechain. Manipulation of the functional group in a second or third coreunit can be effected as shown in the conversion of 57 to 59.

2. Utility of Chemoprotective Polyamines in Regulating Cell Growth andProtecting Against Cancer Therapies

To determine the activity of the described compounds as regulators ofcell growth, as well as to provide mechanistic insight into ways bywhich these compounds regulate cell growth, we examined chemicalinteraction between the subject compounds and nucleic acids and assessedthe extent to which these chemical and growth regulatory propertiesconferred protection in animal tissues against cancer chemotherapy andradiotherapy. Several exemplary compounds of the invention were testedusing various in vitro and in vivo model systems. The subject compoundswere found to inhibit growth of human skin cells at sub-micromolar tomillimolar concentrations, in a manner that could be correlated to theirchemical structure. Consistent with this inhibition of cell growth, thecompounds were shown to bind avidly to helical DNA, to induce expressionof the negative growth regulator, p21, and to block cells within the G1phase of the cell cycle, also in a manner related to their structure.When the subject molecules were applied locally by topicaladministration to rodent skin, they protected the hair follicle cellsand blocked the alopecia normally seen following systemic administrationof a chemotherapy drug.

The in vitro growth inhibitory effects of certain of the chemoprotectivepolyamines of the invention were measured using primary, diploidfibroblasts isolated from human skin. As shown in Table 1, IC₅₀concentrations (the drug concentration that caused a 50% inhibition ofcell growth) for the polyamines ranged from sub-micromolar tomillimolar. TABLE 1 Expt. 1, Expt. 2 Compound # [MW:HCl salt] IC₅₀ (uM)PrC-110 [523.9] 1680 809 PrC-111 [739.0] 980 180 PrC-112 [954.2] 2.53PrC-113 [1169.3] 0.33 0.21 PrC-114 [476.4] 850 240 PrC-115 [691.6] 41001090 PrC-116 [906.7] 5.8 PrC-117 [1121.9] 0.32 0.24 PrC-118 [675.5] 78PrC-119 [725.0] 470 202 PrC-120 [1155.3] 0.22 0.18 PrC-121 [1169.3] 0.15PrC-122 [940.2] 0.81

As part of this invention, heretofore not described in the literature,FIG. 2 shows that the IC₅₀ concentration for each chemoprotectivepolyamine was tightly correlated with the length of the polyamine sidechains (‘arms’) attached to a central butene core, with the long arms,i.e., those containing 16 aliphatic carbon atoms, associated withsub-micromolar IC₅₀ values.

The chemoprotective polyamines of the invention are also able to bind,denature and precipitate DNA from solution As is known in the field, asthe concentration of polyamine is increased, there is a point wherepolyamine binding to helical B-DNA induces single-stranded ‘bubbles’ andconversion to other forms of DNA structure, such as 2-DNA (Feuerstein,B. et al. Nuc. Acids Res. 17:6883-6892, 1989; Basu, H. and Marton, L.Biochem. J. 244:243-246, 1987), as well as precipitating the DNA fromsolution. Table 2 shows that the four molecules that contain ‘16 carbonarms,’ i.e., PrC-113, PrC-117, PrC-120 and PrC-121, all have IC₅₀concentrations that are lower than all of the other molecules thatcontain shorter aliphatic arms. TABLE 2 DNA Bind/ppt. Expt. 1, Expt. 2Compound # [MW:HCl salt] IC₅₀ (uM) PrC-110 [523.9] 270 PrC-111 [739.0]88 PrC-112 [954.2] 93 PrC-113 [1169.3] 35 PrC-114 [476.4] 94 PrC-115[691.6] 37, 83 PrC-116 [906.7] 82, 63 PrC-117 [1121.9] 58, 57 PrC-118[675.5] — PrC-119 [725.0] 89 PrC-120 [1155.3] 57 PrC-121 [1169.3] 49PrC-122 [940.2] 102 PrC-123 [906.7] 131, 117 Spermine 2600This relationship between arm length of the chemoprotective polyamineand increased ability to disrupt and denature B-DNA structure is also aunique aspect of this invention. The increased ability to bind DNA anddisrupt its helical structure may also contribute to the molecule'sability to protect cellular DNA against electrophilic chemotherapy drugmetabolites and against oxygen free radicals generated duringradiotherapy. Both of these toxic modalities are believed to requirenormal B-DNA helical structure within the cell's nuclear DNA in order toachieve chemical or physical disruption of the cellular DNA, the firststep in the apoptotic cascade.

FIG. 3 illustrates that chemoprotective polyamines are able to induceexpression of the negative cell cycle regulatory protein, p21, afterexposing the human skin cells to the polyamine molecules. FIG. 3B showsthat the induced p21 level is greater after a 30 hr exposure compared toa 50 hr exposure to drug. In these experiments, the 23SK human skincells were exposed for 30 hr to an “IC₈₀” dose of each of the indicatedchemoprotective polyamines and then lysed. Cell extracts were thenprepared in order to measure p21 levels by western analysis (FIG. 3A).Results are summarized in Table 3. Although the ability of modifiedpolyamines to induce p21 is known in the literature (Kramer, D. et al.,Cancer Res. 59:1278-1286, 1999), it is a novel aspect of this inventionthat those chemoprotective polyamines with longer aliphatic “arms” werebetter able to induce expression of p21 as shown in FIG. 4. TABLE 3 p21fold-induction Compound # [MW:HCl salt] at IC₈₀ Dose PrC-110 [523.9]2.91 PrC-111 [739.0] 2.22 PrC-112 [954.2] 2.45 PrC-113 [1169.3] 3.21PrC-114 [476.4] — PrC-115 [691.6] — PrC-116 [906.7] 2.32 PrC-117[1121.9] ˜3.0 PrC-118 [675.5] — PrC-119 [725.0] 1.80 PrC-120 [1155.3]2.01 PrC-121 [1169.3] 3.26 PrC-122 [940.2] 1.70 PrC-123 [906.7] 2.22Colcemid 3.31

In FIG. 5, cell histograms showing the results from flow cytometryanalysis of chemoprotective polyamine-treated 23SK skin cells are shown.FIG. 5A shows that for untreated, exponentially growing 23SK cells,59.12% of the cells are present in the S+G2 cell cycle compartments,whereas only 40.88% of the cells are in the G1 compartment. FIG. 5Bshows, as a control treatment, that incubation of cells in serum-freemedium causes a sizable reduction in S+G2 cell compartments (down to5.63% total), and a sizable increase in cells now present in the G1compartment (up to 94.37%). FIG. 5C shows that cells treated withPrC-117 for 72 hr also show a marked reduction in S+G2 compartments(down to 13.77%) and a marked increase in the G1 compartment (up to86.23%). FIG. 5D shows that after the cells treated with PrC-117 for 72hr are switched for 48 hr to medium devoid of the PrC-117 molecule, thedistribution within cell cycle compartments is basically returned tothat seen in cells previously untreated with chemoprotective polyamine(i.e., FIG. 5A). The transient nature of the cell cycle block induced bychemoprotective polyamines is believed to be an important aspect oftheir efficacy, i.e., their ability to block cell cycle progression instem cells during the course of chemo- or radiotherapy, and theresumption of normal stem cell division after a given cancer therapycourse has been completed. Table 4 shows that, of the ninechemoprotective polyamine molecules tested, three caused G1 cell cycleblocks with greater than 75% of the cells present in the G1 compartment,and each of these three molecules contained 16 carbon aliphatic arms.TABLE 4 Cell Cycle Distribution At IC₈₀ Dose (%) Compound # [MW:HClsalt] G1 S G2/M PrC-110 [523.9] 60 26 14 PrC-111 [739.0] 60 25 14PrC-112 [954.2] 61 23 16 PrC-113 [1169.3] 77  8 14 PrC-114 [476.4] —PrC-115 [691.6] — PrC-116 [906.7] 66 11 23 PrC-117 [1121.9] 86 11  3PrC-118 [675.5] — PrC-119 [725.0] — PrC-120 [1155.3] — PrC-121 [1169.3]76  4 20 PrC-122 [940.2] 67 15 18 PrC-123 [906.7] 68 10 21 Colcemid  811 81

Natural polyamines such as spermine, with a 3-4-3 configuration ofaliphatic carbon chains containing terminal amine groups and separatedby intervening amine groups, are known to bind avidly to cellular DNA inthe cell setting. Synthetic polyamines, containing longer aliphaticcarbon segments, typically of four carbons, have been shown to displacenatural polyamines like spermine from DNA because of their greaterbinding affinity for helical DNA. At physiologic pH, each of the aminegroups of a polyamine backbone can protonated to yield an ammoniumcation. Therefore, as the length of a polyamine increases, achieved byoligomerizing a —(CH₂)₄—NH— segment, for example, there are typically anincreased number of ammonium cations distributed along the polyaminebackbone for bonding with anions distributed along the DNA backbone. Asa result, longer, synthetic polyamine analogs compete more effectivelywith spermine in vitro and in vivo for binding to DNA. Binding ofpolyamines to helical DNA has also been shown to confer conformationalchanges to the DNA, such as conversion of helical B DNA to A or Z formsof DNA. And, in vivo, polyamine analogs have also been shown to causecondensation and aggregation of DNA and chromatin within mammalian cells(Basu, H., et al., Cancer Res. 49: 5591, 1989; Basu, H. et al., Biochem.J. 269:329, 1990). Though not intending to be bound by any particulartheory, it is believed that his tight binding and associated distortionof normal helical structure, which is optimized in the design ofchemoprotective polyamines of the present invention, providespharmacologic benefit in at least three ways. First, the pharmacologic,growth inhibitory activity is reversible, as shown, for example, for thePrC-117 molecule in FIG. 5, i.e., by simply stopping topicalapplication, the treated cells are released from growth inhibition thusyielding a ‘transient’ growth regulation. Second, distortion of helicalDNA and the formation of single-stranded bubbles is likely to be thecause, or to be closely related to the cause, of the induced expressionof p21 and the G1 cell cycle block that is associated with its inducedexpression. Third, for many electrophilic, alkylating drugs, reactionwith DNA occurs in two steps, the first step requiring intercalation ofthe drug molecule between nucleoside bases in helical B DNA, and a rapidsecond step involving alkylation of the adjacent DNA base by the drugmolecule. By condensing and altering normal DNA helical form,chemoprotective polyamines are expected to significantly reducealkylation of cellular DNA by electrophilic drugs. Likewise,condensation and alteration of DNA helical form by polyamine binding invitro has also been shown to dramatically reduce the number of singlestrand breaks induced when the DNA is directly irradiated in vitro(Spotheim, M., lit. J. Radiat. Biol. 68: 571-577, 1995).

When comparing chemoprotective polyamines to those polyamine analogsthat have been previously described, there are a number of markeddifferences. For instance, Edwards (U.S. Pat. Nos. 5,217,964 and5,434,145) attached one or more alkyl-thiophosphate or alkyl-thiolgroups to one or more of the backbone amines of short aliphaticpolyamines, or to one or more backbone amines as well as to one or moreterminal benzyl groups on equally short polyamines. In comparison, thepresent inventors have designed and synthesized chemoprotectivepolyamine molecules which: i) optimize both the polyamine side chain(“arm”) length and overall molecule length to achieve tight DNA binding,ii) project or “display” a protective functional group physically awayfrom the DNA to which the chemoprotective polyamine is strongly bound,iii) attach the functional group to a polyamine backbone carbon atominstead of to one of the backbone amine groups, iv) in certainembodiments, display functional groups from allylic positions ofolefinic core segments that are present in chemoprotective polyamines;this is done by design to enhance reactivity of the group, v) include arange of functional groups that are “displayed,” including —SH, —OH,—NH₂, —NHR, —NR₂, —SH and —SCH₃ moieties, singly or in combination, aswell as other groups that are known to vary in their degree ofnucleophilicity or ability to scavenge free radicals, vi) include thedisplay of more than one functional group per polyamine molecule, andvii) in some embodiments, include a rigid platform from which thefunctional group is projected or displayed on a spacer aliphatic chainaway from the DNA in a manner that better enables the “sentinel group”to scavenge or trap electrophiles/oxygen radicals from the cellularmilieu before they attack other known nucleophilic groups within DNA,such as the 2-amino group of deoxyguanosine.

This ability to scavenge and trap chemical/physical reactants within acell does not require the chemoprotective polyamine to be physicallyattached to cellular DNA or RNA. Rather, simple molar presence of suchnucleophilic or other protective functional groups in cells would beexpected to be protective. For instance, previous work in the field hasshown a positive, linear correlation between the intracellularconcentration of the physiologic nucleophile, glutathione (GSH), and theconcentration of an electrophile required to kill the exposed cells (Ho,D. and Fahl, W. E., J. Biol. Chem. 259: 11231-11235, 1984). In anothermechanism by which chemoprotective polyamines might protect cellsagainst cytotoxic threats, they may serve as a “stealth” vehicle bywhich to load cells with —SH or other nucleophilic or protective groups.Whereas, it is well known in the field (Levy, E. et al., Proc. Natl.Acad. Sci. USA 90:9171-9175, 1993) that the SH-containing nucleophile,glutathione, is not taken up by cells in a physiologic setting, the cellmembrane polyamine transporter (PTS), which is known to mediate theuptake of polyamines, molecules containing multiple charged sites,should efficiently transport functional group-displaying polyamines intocells, and that this would provide an efficient means to “load” cellswith, e.g., an SH-containing polyamine, which could serve as aglutathione surrogate. Once loaded with the polyamine, these cells wouldbe protected from subsequent toxic challenges, such as those seen withtransient chemotherapy and radiotherapy regimens. The results in thetables and figures that show the same growth regulating efficacy foreach of the SH-containing chemoprotective polyamines (i.e., PrC-114,PrC-115, PrC-116, PrC-117) as for those chemoprotective polyamineswithout SH groups implies that the SH-containing molecules aretransported into the human fibroblasts equally well, and that they bindwith equal affinity to cellular DNA. Moreover, the fact that each of theSH-displaying chemoprotective polyamines exemplified herein has shownprotective activity in the rat cytoxan-induced alopecia assaydemonstrates that the displayed nucleophile is also active within thecell milieu.

Another way to increase the molar presence of nucleophiles/scavengerswithin the nuclear environs is to display more than one such functionalgroup on each chemoprotective polyamine molecule. In embodiments wheretwo —SH groups are displayed on a single polyamine, then a reducingagent such as sodium borohydride or others as known in the art may addedto the pharmaceutical preparation to reduce any —S—S— disulfide bondsthat might be formed when —SH groups are present in an oxygen containingmedium. An alternate strategy to avoid disulfide bond formation is to“cap” the displayed sulfur atom with a CH₃ group to prevent interactionof the sulfur atoms, while still retaining the capacity of the sulfuratom to scavenge electrophiles/oxygen radicals.

The use and placement of protective functional groups on the backbone ofchemoprotective polyamines is also significantly different from theattachment of —CH₂CH₂SPO₃H₂ or —CH₂CH₂SH groups to polyamines describedby Edwards in U.S. Pat. Nos. 5,434,145 and 5,217,964. In U.S. Pat. No.5,434,145, Edwards showed bonding of alkyl-thiophosphate or alkyl-thiolgroups to one or more of the 3-4 backbone amines present in the shortpolyamine molecules. By modifying the secondary amines in the polyaminebackbone with alkyl-thiophosphate groups, the amines were converted totertiary amines, which markedly alters the basicity of the individualmodified amine, as well as the overall polyamine molecule. Theattenuated basicity of the individual amine groups is accompanied by analteration in 3-dimensional structure at these sites. With added alkylfunctionality on the amine nitrogen atoms, steric bulkiness increases,so the ability or freedom of the molecule to rotate and twist at thesesites is markedly reduced. The altered basicity and steric constraintsin these short spermine-like polyamines perturbs DNA binding by thepolyamine as compared to their natural polyamine counterparts. Given thealready very high IC₅₀ concentration of spermine for DNAbinding/precipitation (nearly 1,000-fold higher than for mostchemoprotective polyamines; see Table 2), it is possible that themodification of backbone amines described by Edwards would eliminate DNAbinding altogether in cells at the concentrations of drug that could bepharmacologically achieved. The attenuated basicity of theamine-modified polyamine molecules in Edwards could also affect theirpharmacologic delivery characteristics. In topical applications to skinand other epithelial surfaces, there is an accepted relationship betweenthe degree of ionization at physiologic pH of an applied drug and thedegree to which it permeates or traverses the surface cells. In contrastto Edwards, the functional group used in the chemoprotective polyaminesof the invention, whether —SH or one of several other groups (e.g., OH,N-ethyl, N-methyl, N-dimethyl; see FIG. 1), is bound to a carbon atomwithin the polyamine backbone. This was done specifically to avoidperturbing the DNA binding characteristics of each of the backbone aminegroups, while still achieving the display of reactive functional groups.

In U.S. Pat. No. 5,217,964, Edwards discloses the linking of one or morealkyl-thiophosphate or alkyl-thiol groups to the polyamine backbonethrough one or more terminal benzyl group(s) or through one or more ofthe backbone amine groups. Work within the field (Huber, M., J. Biol.Chem. 271:27556-27563, 1996) has shown that polyamines containing one ormore aromatic groups are well-suited to serve as inhibitors of themembrane polyamine uptake transporter, and predictably, they themselvesare not taken up into cells. Consistent with the above observations,Edwards provides no information regarding biological activity for any ofthe structures proposed in U.S. Pat. No. 5,217,964 or U.S. Pat. No.5,434,145.

FIGS. 6A-6E illustrate the efficacy of each of the indicatedchemoprotective polyamines in protecting against Cytoxan-inducedalopecia in the rat model Hussein et al., 1990, infra). In this protocol(See Example 2), chemoprotective polyamines are applied topically to therat pups' backs in an alcohol:water delivery vehicle, once per day, forfive days before and five days after a single systemic dose of Cytoxan.As seen, topical chemoprotective polyamines conferred significantprotection against the generalized alopecia that was seen to occur inthe vehicle-treated rat pups.

3. Topical or Local Administration of Pharmaceutical Preparations

As described above, the chemoprotective polyamines of the presentinvention have been shown to inhibit the growth of normal human skincells, to modify normal B-DNA helical structure, to induce expression ofthe negative cell cycle regulator, p21, to cause a G1-specific cellcycle block, and to protect against chemotherapy-induced alopecia anddermatitis in an animal model. Thus, the compounds of the invention areparticularly suitable for treatment of humans to prevent the local sideeffects of cancer chemotherapy and radiotherapy. Based upon their growthregulatory effects, chemoprotective polyamines may also find utility inother applications where inhibition of cell growth would beadvantageous, including regulating proliferative conditions of the skin,such as psoriasis and dermal nevus.

Two important targets for delivery of such protective therapies are (1)the epithelial cells of the skin, including hair follicles and theepidermis, and (2) the epithelial cells lining the oral and entiregastrointestinal (G1) or urogenital tract The method of protection ofthese tissues with chemoprotective polyamine comprises administering toa population of epithelial cells a composition consisting of achemoprotective polyamine and a delivery vehicle for a time and in anamount effective to protect the non-neoplastic cells from damage duringthe cancer chemotherapy or radiotherapy. In one embodiment, the methodis used to prevent alopecia during cancer therapy, by topically applyingthe composition to the scalp. In another embodiment, the method is usedto prevent gastrointestinal distress due to cancer therapy byadministering the composition orally. In another embodiment, the methodis used to prevent mucositis from chemotherapy or radiotherapy byadministering the composition topically to the appropriate region of thebody. In yet another embodiment, the method is used to preventradiation-induced dermatitis, skin rash, and ulceration at the site ofirradiation by applying the composition to the skin.

Administration of chemoprotective polyamines to human or non-humansubjects can be achieved in several ways. The preferred administrationroute is topical, to tissue sites including the skin, as well asoropharyngeal and gastrointestinal mucosal surfaces. It can also bedelivered locally to an internal organ, tissue or regions thereof. Itshould be noted, as with all pharmaceuticals, the concentration andtotal amount of polyamine administered will vary depending upon thetissue being treated, the mode of administration, the size and conditionof the subject being treated, and the particular chemoprotectivepolyamine being used.

Compositions of chemoprotective polyamines formulated in deliveryvehicles are well-suited to be administered topically to the skin orsurfaces of the mouth, GI or urogenital tract. Pharmacologicconcentrations of chemoprotective polyamines can protect normal,non-neoplastic cells from cancer therapy-associated cell damage. Byproducing a local gradient effect within the tissues, the topicallyapplied polyamine produces a local protective effect at the intendedregion. This dose-dependant gradient of topical drug can effectivelyprotect normal proliferating cells rendering them less susceptible toradiation or chemotherapy. Importantly, while this local effect wouldprotect normal cells, in contrast, any deeper-seated tumor cells wouldbe less affected by the topical polyamine composition, and would remainsensitive to the cancer therapeutic. Moreover, topical delivery of achemoprotective polyamine, which has a highly positive charge atphysiologic pH, should diminish any systemic exposure and limit theeffect on any tumor cells or normal host organ cells. Given the hosttoxicity that has been previously observed when polyamine analogs wereadministered systemically (Creaven, P. et al., Invest. New Drugs15:227-234, 1997; Streiff, R and Bender, J., Invest New Drugs 19:29-39,2001), this provides another important reason to avoid systemic deliveryof the chemoprotective polyamine molecules. The intended protection ofnormal tissue is achieved by an appropriate formulation ofchemoprotective polyamine in combination with an appropriate deliveryvehicle depending on the administration site (e.g. dermal/intradermal ormucosal). A pharmaceutical composition comprising a chemoprotectivepolyamine formulated with an appropriate delivery vehicle will haveutility in any normal cell type susceptible to the side effects ofcancer therapy that is accessible by topical delivery.

Thus, the chemoprotective polyamines of the invention are administeredtopically (or locally) to protect patients from the side effects ofcancer therapy. The term “topical” denotes the administration of a drugintended to act locally rather than systemically. In the presentinvention, “topical” or “local” delivery is directed to epidermal anddermal cells of the skin and scalp (including cells lining hairfollicles), as well as mucosal cells of the mouth, salivary glands,throat, gastrointestinal system and urogenital tract. For some of theselatter locations, compositions may be formulated for oral or nasaldelivery, or as suppositories. The goal of such delivery systems is tocontact these internal surfaces topically with the polyamine effectors.

The local delivery of drug molecules within the skin or mucous membranesusing a noninvasive delivery system has many attractions, includingpatient acceptability due to the noninvasiveness of the procedure,avoidance of gastrointestinal digestion and disturbances, and first-passmetabolism of the delivered molecule. Topical delivery is not anefficient means for systemic drug delivery. It is estimated that onlybetween 1%-15% of a drug in most topical formulations is systemicallybioavailable. In preferred embodiments of the invention, less than 10%,preferably less than 5% and most preferably less than 1% of thepolyamine effector, provided topically e.g., dermal, intradermal,mucosal or G1 epithelial delivery, move to reach the dermis and/or otherunderlying tissues.

Topical delivery vehicles can take the form of aqueous oraqueous:alcohol solutions, emulsions, creams, lotions, ointments, gelsor liposomes.

Solutions are the most traditional types of formulations for topicaldermal drugs, where the agent is solubilized in a solvent Solvent-basedsystems are simple and effective constituents of topical deliveryvehicles for some drugs. Alcohols are the most commonly used solventsfor topical solutions. Typically, the drug is combined into a water andalcohol mixture. The alcohol content varies between 10-100%. Alcoholsused include ethanol, propylene glycol, polyethylene glycols, methanol,or butanediol. Each of these types of alcohols is suitable for use inthe present invention; others not listed are also suitable, as would beunderstood by one of skill in the art. High alcohol content solutionssuch as solutions of 70% ethanol in water or ones containing 60%ethanol, 20% propylene glycol and 20% water, are particularly good atpenetrating the stratum corneum of the epidermis. Topical minoxidil, ahair regrowth treatment, uses the latter formulation as the deliveryvehicle.

Solution-based delivery systems are particularly suitable for thedelivery of small organic molecules. In a preferred embodiment of theinvention, particularly for administration of chemoprotective polyaminesto the epidermis, alcoholic solutions, as described above, are utilized.An aqueous alcohol-based delivery vehicle has been proven to be highlyeffective for topical administration of chemoprotective polyamines.Advantages of this delivery system include, ease of manufacturing, easeof application, fast drying, lack of residue on skin, and ease ofanalysis of active drug compound after formulation. Solution typeformulations are typically administered using dropper bottles or asaerosols.

Emulsions form the basis of cream and lotion-type formulations.Typically, these formulations are colloidal dispersions composed of twoimmiscible phases; an oil phase and an aqueous phase with an emulsifier.Typical oils used in emulsions include stearyl alcohol, isopropyllanolate, isopropyl myristate, cetyl alcohol, and vitamin E. Emulsifiersare essentially surfactants that lower the surface tension of theimmiscible phases. Most emulsifiers tend to be fatty acid esters orstearates of glycerol, sorbitan, or polyoxyethylene (POE). Depending onthe location of the oil and water, emulsions are oil-in-water,water-in-oil or combinations thereof. The preparation of an emulsioncommonly requires some mechanical shear force with heat to mix theinternal and external phases. Most topical emulsions contain viscositybuilders such as natural gums (alginates, carrageenan, tragacanth,pectin, xanthan or collagen) at 1-5% to thicken the preparation. Higherpercentages of viscosity builders produce creams, a lower percentageform lotions. Complete formulations for emulsions (creams and lotions)generally include water, alcohol, propylene glycol, sodium laurylsulfate and white wax. In alternative formulations, they include water,alcohol, glycerol, phosphatidyl choline, lysophosphatidyl choline andtriglycerides. For administration of chemoprotective polyamines to theepidermis, emulsions are particularly well suited. Ease ofadministration, good local retention and slow release of drug are someof the attractive characteristics of emulsions for a topical deliverysystem.

Ointments are composed of fluid hydrocarbons meshed in a matrix ofhigher melting solid hydrocarbons. The hydrocarbon ointment base istypically petrolatum and white ointment Ointments are prepared bymelting the base, followed by the addition of excipients, such asantioxidants to the fluid. The drug is then suspended into the ointmentby milling. Due to the high oil content, ointments tend to be greasy.Adding components, such as microcrystalline cellulose, which gives theointment a dry feel on the skin, can reduce greasiness. All ingredientslisted above for preparation of ointments are suitable for use in thepresent invention, as well as unlisted ingredients typically employedfor such purpose by one of skill in the art.

Gels are semisolids consisting of a gelling agent that is penetratedwith liquid solvent. The concentration and the molecular weight of thegelling agent affect the consistency of vehicle formulation. The gellingagent is a suspension of either large organic or small inorganicmolecules. The large organic molecules consisting of either natural orsynthetic polymers exist as randomly coiled chains that entangle andform the gel structure. Some common polymers of this kind are naturalgums, cellulose derivatives and acrylic acid polymers. Another class ofthese gels, called thermally sensitive gels, is prepared frompoloxamers. In contrast, the small inorganic molecules form the gelstructure by forming a somewhat organized three-dimensional network.Common small inorganic polymers include colloidal solids found in silicaand clays. The nature of the solvent determines whether the gel is ahydrogel (water-based) or an organogel (non-aqueous solvent based). Gelsare attractive topical delivery vehicles for chemoprotective polyaminesbecause they are relatively easy to prepare and tend to have a longresidence time at the site of application allowing the slow release ofcompound at the desired site. All ingredients listed above forpreparation of gels are suitable for use in the present invention, aswell as unlisted ingredients typically employed by one skilled in theart for such purpose.

Liposomes are vesicles consisting of amphipathic lipids arranged in oneor more concentric bilayers. When lipids are placed in aqueous medium,the hydrophilic interaction of the lipid head groups with water resultsin the formation of multilamellar and unilamellar systems or vesicleswhich resemble biological membranes in the form of a spherical shell.Liposomes may be small (0.025-0.05 um) to large multilamellar vesicles(0.05-10 um). Lipids used to prepare the liposomes includephospholipids, sphingolipids, glycosphingolipids, saturated glycerides,steroids (e.g., cholesterol) and synthetic phospholipids. Liposomes aretypically prepared by melting the lipid together in aqueous solvent withan emulsifier like POE. The drug is then added and the liposomes aregenerated through mixing or sonication. The drug is usually entrapped inthe vesicle structure. These basic liposomes are sometimes referred toas “conventional liposomes.” Several other types of liposomalpreparations exist including (1) sterically stabilized liposomes, whichare surface coated with an inert hydrophilic polymer, such aspolyethylene glycol; (2) targeted liposomes, to which are attachedtargeting ligands, such as antibodies or fragments thereof, lectins,oligosaccharides or peptides (e.g., choleratoxin B (CTB) is used totarget liposomes to the gastrointestinal epithelium); and (3) reactiveor “polymorphic” liposomes, which change their phase and structure inresponse to a particular interaction (this group includes liposomessensitive to ions (pH, cations), heat and light, among other stimuli.

Liposomes are good vehicles for dermatological applications. Liposomaldelivery offers certain advantages over more conventional formulations,including: (1) reduced serious side effects and incompatability fromundesirably high systemic absorption; (2) significantly enhancedaccumulation of the delivered substance at the site of administrationdue to high compatability of liposomes with stratum corneum; (3) readyincorporation of a wide variety of hydrophilic and hydrophobic moleculesinto the skin; (4) protection of the entrapped compound from metabolicdegradation; and (5) close resemblance to the natural membrane structureand their associated biocompatibility and biodegradability. Allingredients listed above and for preparation of various types ofliposomes are suitable for use in the present invention, as well as anyother such ingredients typically employed by one skilled in the art forsuch purpose.

In order to achieve efficient delivery of a chemoprotective polyamineinto the skin, one embodiment of the invention includes variousformulations of liposomes (phospholipid-based vesicles, cationicliposomes, nonionic liposomes, non ionic/cationic liposomes, pegylatedliposomes, PINC polymer, and propylene glycol and ethanol mixture(commonly used vehicle for administering minoxidil), and nonionicliposome/propylene glycol and ethanol mixtures. Reactive liposomes maybe preferred for other embodiments of the present invention. Inclusionof cationic amphiphiles as a minor component of liposomes facilitatesthe association with negatively charged solutes, the rapid binding ofliposomes to the cell surface, and the cellular uptake of liposomes.pH-sensitive liposomes have been developed to improve the efficiency ofthe cytoplasmic delivery of antitumor drugs, proteins, and nucleicacids. Most pH-sensitive liposomes have been prepared usingphosphatidylethanolamine (PE). PE alone does not form liposomes and isprone to form the inverted hexagonal phase (HI). However, liposomes canbe prepared by adding another bilayer-stabilizing, amphiphilic lipidcomponent to PE. Titratable amphiphiles having a carboxyl group havebeen used as a component for the preparation of pH-sensitive liposomes.Because the ability to stabilize a bilayer membrane by these titratableamphiphiles decreases under acidic conditions, destabilization resultsin the fusion of the liposomes. pH-sensitive liposomes are stable atphysiological pH, and are internalized by cells through an endocyticpathway, which exposes the liposomes to an acidic pH. Liposomes withinthe endosome are destabilized and possibly fuse with the endosomemembrane, resulting in release of their contents into the cytoplasmwithout degradation by lysosomal enzymes.

In other embodiments of the invention, sterically stabilized, inertliposomes are particularly suitable. In still other embodiments,targeted liposomes may be used to advantage.

For many applications, mucosal delivery will be used for delivery ofchemoprotective polyamines. Mucosal delivery defined here is the localdelivery of polyamine effectors to the mucosa of the mouth, GI, andurogenital tract. Mucosally active drugs, can be formulated as eithersolutions, emulsions or creams, ointments, gels or liposomes using theingredients described above. In addition, there are also specialexcipients specifically designed for mucosal delivery. The description,composition, and applicability of these major types of mucosal deliveryforms are set forth below. Each is considered suitable for practice ofvarious embodiments of the present invention.

In general, the structure of the mucosal surface is composed of anoutermost layer of stratified squamous epithelium, below which lie abasement membrane, a lamina propria followed by the submucosa as theinner-most layer. The mucosae of areas subject to mechanical stress suchas the gingivae or the hard palate are also keratinzed, similar to theepidermis. Depending on the keratinition, the mucosa is somewhatpermeable. The permeability of oral mucosa is 4-4000 times greater thanthat of the skin Permeability of intestinal mucosa is even greater. Thecells of the epithelia are surrounded by an intercellular groundsubstance, mucous, the principal components of which are complexes ofproteins, carbohydrates, lipids and ceramides. Primarily, specialmucous-secreting cells, called goblet cells, synthesize mucous. However,in the oral mucosa, most of the mucous is produced by the major andminor salivary glands. Mucous forms a strongly cohesive gel structurethat will bind to the epithelial cell surface as a gelatinous layer. Thepenetration of this mucous layer and the local retention of compoundbecause of its permeability must be achieved for effective mucosal drugdelivery. However, this route of administration is very important forthe delivery of compounds designed to protect mucosal surfaces fromcancer therapy. Since the mucosal surface is a common site in which manyof the unwanted side effects occur, the use of formulatedmucosally-active drugs designed to prevent these effects is warranted.

Issues to be considered with mucosal delivery are (1) low flux or drugtransport through the mucous layer and (2) poor retention andbioadhesion at the mucosal site. Mucosal permeation enhancers aredesigned to improve drug flux or penetration at the mucosal surface. Theuse of these enhancers can increase drug permeability by 100-fold ormore. Various permeation/absorption enhancers vary in molecular weightand physicochemical properties. In a preferred embodiment for mucosaldelivery, permeation enhancers are included in formulations for deliveryof chemoprotective polyamines to the mucosal surface. Most types ofenhancers are detergents that include: sodium glycocholate, sodiumtaurocholate, polysorbate 80, sodium lauryl sulfate, lauric acid, andvarious alkyl glycosides. Other examples of enhancers include: dextrins(cyclodextrin, dextran sulfate), fatty acids (phosphatidylcholine,lysophosphatidylcholine), heterocyclic compounds (azone), and smallmolecules (benzalkonium chloride, cetyltrimethylammonium bromide). Eachis contemplated for use in the present invention as are other unlistedingredients typically used for such purpose, as would be appreciated byone of skill in the art

The addition of mucoadhesives to the formulation can improve localretention of mucosally delivered compounds. In another preferredembodiment for mucosal delivery, mucoadhesives are included in thepolyamine effector formulations of the invention. Mucoadhesive compoundsare primarily synthetic or natural polymers that can adhere to the wetmucosal surface. These include synthetic polymers such as monomericalpha cyanoacrylate, polyacrylic acid, hydroxypropyl methylcellulose,and poly methacrylate derivatives. Glue-like polymers include epoxyresins and polyurethanes. Naturally occurring mucoadhesives includechitosan, hyaluronic acid and xanthan gum. Each is contemplated for usein the present invention as are other unlisted ingredients typicallyused for such purpose, as would be appreciated by one of skill in theart.

Other delivery vehicles are also suitable for use in the presentinvention, particularly for administration of polyamine effectors to themucosa and lumen of the GI and urogenital tract. Nonlimiting examplesinclude: (1) oils such as vegetable oils or fish oils (which can beencapsulated into standard gel capsules); and (2) emulsions prepared,for example, by dispersing polyoxyethylene ethers, e.g., 10-stearylether (Brij 76) in aqueous buffer.

Other examples of delivery vehicles suitable for the GI or urogenitalmucosa include biodegradable microparticles (preferably in the range of0.1-10 uM diameter) of polylactic polyglycolic acid, which have beenused to deliver proteins to Caco-2 cells as an in vitro model system forgastrointestinal uptake via oral drug delivery (Desai et al., Pharm.Res. 14: 1568-1573, 1997). Significant uptake of proteins carried bypolystyrene particles into cells lining the small intestine of the rathas been demonstrated (Hillery et al., J. Drug Targeting 2: 151-156,1994). Indeed, delivery of protein-containing microparticles has beenreported from the GI lumen all the way to the submucosal vasculature(Aphrandan et al., Biol. Cell 61: 69-76, 1987). Therefore, suchpolymeric microparticles are quite suitable for oral delivery ofpolyamine effectors to gastrointestinal epithelial cells, which arefound on the surface of the GI lumen.

Thus, chemoprotective polyamines are formulated as pharmaceuticalpreparations for topical or local administration to patients. Thefollowing sites of local administration of these pharmaceuticalpreparations are contemplated: oral, nasal, ophthalmic,gastrointestinal, urogenital and dermal (cutaneous). The term “patient”or “subject” as used herein refers to human or animal subjects (animalsbeing particularly useful as models for clinical efficacy of aparticular composition). Selection of a suitable pharmaceuticalpreparation depends upon the method of administration chosen, and may bemade according to protocols well known to medicinal chemists.

The pharmaceutical preparation comprising the compositions of theinvention are conveniently formulated for administration with abiologically acceptable medium such as water, buffered saline, alcohols,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol and the like), dimethyl sulfoxide (DMSO), oils, detergents,suspending agents or suitable mixtures thereof, as compatible with thespecific delivery vehicles described above. The concentration of aparticular composition in the chosen medium will depend on thehydrophobic or hydrophilic nature of the medium, in combination with thespecific properties of the delivery vehicle and active agents disposedtherein. As used herein, “biologically acceptable” or “pharmaceuticallyacceptable” refers to those compounds, materials, compositions, and/ordosage forms that are, within the scope of sound medical judgment,suitable for contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem complications commensurate with a reasonable benefit/risk ratio.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like. Thus, the term“acid addition salt” refers to the corresponding salt derivative of aparent compound Eat has been prepared by the addition of an acid. Thepharmaceutically acceptable salts include the conventional salts or thequaternary ammonium salts of the parent compound formed, for example,from inorganic or organic acids. For example, such conventional saltsinclude, but are not limited to, those derived from inorganic acids suchas hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric andthe like; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like. Certain acidic or basic compounds of the present inventionmay exist as zwitterions. All forms of the compounds, including freeacid, free base, and zwitterions, are contemplated to be within thescope of the present invention.

The topical formulation can contain a variety of excipients thatfunction to stabilize and solubilize the drug formulation, increasepermeation, and protect and aid in the application to the skin. Oil orwater-based excipients are primarily added to improve drug solubilityand spreadibility to the formulation. Surfactants may be added totopical formulations as detergents, solubilizers, emulsifiers, andwetting agents.

It will also be appreciated by persons of skill in the art thatpharmaceutical formulations of the invention may contain more than onechemoprotective polyamine. Various combinations of such agents may beuseful for certain applications, and formulations of such combinationswould be prepared according to the general guidelines set forth above.Moreover, one or more chemoprotective polyamines may be combined withother agents, such as other anti-proliferative agents or chemoprotectivedrugs, to provide a pharmaceutical formulation that is effective by twodifferent modes of action. An anti-proliferative agent suitable for suchuse is the cyclin-dependent kinase II inhibitor described in PCTapplication US00/05186, published Dec. 28, 2000 as WO 00/78289 orgenistein, an inhibitor of tyrosine protein kinase. A chemoprotectiveagent suitable for such use is resveratrol (trihydroxy-trans-stilbene).Several classes of “chemoprotective inducing agents” (agents that inducethe cell's endogenous defense processes) that may be combined with thechemoprotective polyamines of the invention are described in detail incommonly-owned, co-pending U.S. application Ser. No. 09/565,714, filedMay 5, 2000, and International Application No. PCT US01/14464, filed May4, 2001, the entireties of each of which are incorporated by referenceherein. Further, certain of those chemoprotective inducing agents alsopossess anti-proliferative activity.

The pharmaceutical preparation is formulated in dosage unit form forease of administration and uniformity of dosage. Dosage unit form, asused herein, refers to a physically discrete unit of the pharmaceuticalpreparation appropriate for the patient undergoing treatment Each dosageshould contain a quantity of the chemoprotective polyamine calculated toproduce the desired protective effect in association with the selectedpharmaceutical carrier. Procedures for determining the appropriatedosage unit are well known to those skilled in the art. As used herein,“therapeutically effective amount” refers to an amount of a compound asdescribed herein that may be effective to inhibit, or treat the symptomsof particular disorder or side effect. The term “prophylacticallyeffective amount” refers to an amount of a compound as described hereinthat may be effective to prevent, inhibit, or diminish the onset thesymptoms of a particular disorder or side effect.

Dosage units may be proportionately increased or decreased based on theheight and weight of the patient Appropriate concentrations forachieving protection of a target cell population or tissue from thetoxic effect of a particular chemotherapeutic agent may be determined bydosage concentration curve calculations, as known in the art

As one example, for topical applications, the chemoprotective polyaminemay be used at concentrations ranging from 1-100 mM in an appropriatecarrier (e.g., alcohol solvent) applied to the scalp or other dermalsite. This dosage is arrived at from results of experiments using arodent model and the range of dosages is a function of results obtainedfrom experiments using several different molecules that ranged in doseeffectiveness. The volume of material applied to the skin ranges by sizeof surface area to be covered; e.g., scalp treatment for young childrenrequiring 3-5 ml, the amount being increased in adults to 10-20 ml perapplication.

As another example, for gastrointestinal administration, the oral doseof the chemoprotective polyamine in an appropriate medium (e.g.,solvent, liposome emulsion) is normalized to the lumenal surface area ofthe stomach and duodenum. This would assume that the patient consumesthe material on an empty stomach upon rising in the morning.

The pharmaceutical preparation comprising the compositions of theinvention may be administered at appropriate intervals, before, during,or after a regimen of chemotherapy and/or radiotherapy. The appropriateinterval in a particular case would normally depend on the nature of thechemotherapy or radiotherapy and the cell population targeted forprotection.

For instance, for prevention of chemotherapy-induced alopecia, solvents,liposomes or other delivery vehicles containing the chemoprotectivepolyamine can be further formulated to be delivered, (e.g., as a topicalcream, or gel) to the scalp of a patient prior to scheduledadministration of chemotherapy. By protecting the epithelial cells thatline the exposed surface of hair follicles from the chemotherapy drug,the loss of hair commonly associated with cancer chemotherapy isprevented. Likewise, for the treatment of radiation-induced dermatitis,the chemoprotective polyamine can be further formulated as a gel,ointment or cream containing moisturizers. This would further protectthe epidermis from radiation damage. The topical formulation preferablyis initiated several days prior to the cancer therapy, to ensure thatthe epithelial and mucosal cells are adequately treated. The formulationmay then continue to be applied during the course of chemotherapy.

For protection of the gastrointestinal epithelium, the chemoprotectivepolyamine is formulated to be delivered by mouth to a patient prior toscheduled administration of cancer therapy. Administration of theprotective formulation in the 1-5 days prior to radiotherapy or theinfusion of the chemotherapeutic agent thus confers protection tosusceptible mucosal epithelial cells. For example, the patient would beinstructed to consume a “shake” containing the chemoprotective polyaminein an orally acceptable solution or liposome emulsion before breakfastin the morning, in the 1-5 days preceding chemotherapy. This would allowthe chemoprotective polyamine to be present when the chemotherapy drugsor radiotherapy act on the GI mucosal epithelium.

The examples that follow are included to aid in a more completeunderstanding of the present invention. The examples do not limit theinvention disclosed and claimed herein in any fashion. Referencenumerals are to the reaction schemes described above. All purificationcolumns were carried out using silica gel (230-400 mesh) with eluantnoted. Silica gel plates (250 micron) were used for all thin layerchromatography (TLC) with the appropriate solvent system noted.

EXAMPLE 1 Preparation of compounds used in Synthetic Schemes

Scheme 1:

Compound 2: 2 M ethylamine (compound 1) in tetrahydrofuran was stirredin a pressure bottle at <0° C. and mesitylene sulfonyl chloride (3 molarequivalents wrt ethylamine) was added in portions so that thetemperature did not exceed 10° C. Dichloromethane and triethylamine wereadded and the pressure bottle sealed. The reaction was stirred in a 30°C. water bath for one hour and at RT for 30 minutes. The reactionprogress was monitored by TLC using 8:2 heptanes: ethyl acetate as themobile phase. Water was added and the organic layer was separated, thewater layer was extracted once with dichloromethane, the combinedorganic layers were washed twice with water and condensed under vacuum.The product was used without further purification.

Compound 3: NaH (1.2 molar equivalents wrt compound A) was stirred,under N₂, at 10° C. and dimethylformamide was added. Compound 2dissolved in tetrahydrofuran was added and stirred until the evolutionof H₂ gas ceased. Bromobutyl (or any N-alkyl depending on desireddistance between amines)-phthalimide (1.1 molar equivalents wrt tocompound 2) was added in one portion and NaI was added. The reaction washeated to 60° C. and the progress monitored after several hours by TLCusing 7:3 heptanes: ethyl acetate as the mobile phase. The reactioncontents were condensed under vacuum and dissolved in ethyl acetate andwater. The organic layer was separated, the aqueous layer was extractedwith ethyl acetate, and the combined organic layers were washed withdilute brine and condensed under vacuum. The product was used withoutfurther purification.

Compound 4: Ethanol was heated to 70° C. and compound B dissolved in hotethanol was added. Hydrazine hydrate (2.5 molar equivalents wrt compound3) was added all at once and the reaction was stirred at 70° C.overnight The reaction progress was monitored by TLC using 6:4 heptanes:ethyl acetate as the mobile phase. The completed reaction was cooled onice and a white precipitate formed. The precipitate was removed byfiltration and the filtrate condensed under vacuum. The resultingsemisolid was dissolved in dichloromethane and water. The organic layerwas separated, the aqueous layer was extracted with dichloromethane andthe combined organic layers were washed with water and condensed undervacuum. The product was purified by column chromatography using silicagel and 90:9:1 dichloromethane: methanol: ammonium hydroxide as theeluant

Compound 5: Mesitylene sulfonyl chloride (1.1 molar equivalents wrtcompound 4) dissolved in dichloromethane was stirred, under N₂, at 10°C. Compound C dissolved in dichloromethane was slowly added so that thetemperature did not exceed 15° C. The reaction was cooled to 10° C. andtriethylamine (1.2 molar equivalents wrt compound 4) was added. Thereaction was stirred at RT for several hours. The progress was monitoredby TLC using 1:1 heptanes: ethyl acetate as the mobile phase. Thereaction was quenched by adding water and stirring for 20 minutes. Theorganic layer was separated, the aqueous layer was extracted with ethylacetate then dichloromethane, and the combined organic layers werewashed with water and condensed under vacuum. The product was purifiedby column chromatography using silica gel and 6:4 heptanes: ethylacetate as the eluant

The polyamine side chains are elongated by repeating steps 2-4 until thedesired length is reached.

Scheme 2:

Compound 16: Dihydroxyacetone dimer, compound 15, was stirred indimethylformamide, under N₂, at 2° C. Imidazole (5.02 molar equivalentswrt. Compound 15) then tert-butyl dimethylsilyl chloride (4.99 molarequivalents wrt compound 15) were added. The reaction was stirred at RTfor 2 hours. Ice water was added and the reaction stirred for 20minutes. The organic layer was separated, the aqueous layer exacted twotimes with ethyl acetate, the combined organic fractions were washedwith dilute brine, dried over anhydrous MgSO4, filtered, and condensedunder vacuum to yield brown oil. The oil was purified by columnchromatography using silica gel and 97:3 heptanes: ethyl acetate then95:5 heptanes: ethyl acetate as the eluant

Compound 17: NaH (1.1 molar equivalents wrt compound 1) was stirred,under N₂, in an ice bath and toluene was added. Triethylphosphonoacetate (1.01 molar equivalents wrt compound 16) was slowlyadded so that the temperature did not exceed 110° C. The reaction wasstirred on ice until all observed effervescence stopped. The reactionwas removed from the ice bath and compound 16 (bis-OTBS acetone) wasadded dropwise. The reaction was stirred at RT for 1.5 hours and ethanolwas added to dissolve a precipitate that had formed. Water was added toquench the reaction. The organic layer was separated, the aqueous layerextracted once with ethyl acetate, and the combined organic layers werewashed with brine and dried over anhydrous MgSO₄. The organic solutionwas filtered and condensed under vacuum to yield yellow oil. The oil waspurified by column chromatography using silica gel and 98:2 heptanes:ethyl acetate.

Compound 18: Compound 2 was stirred in ether and cooled, under N₂, to−80° C. in an acetone/dry ice bath. Diisobutyl aluminum hydride (1.5molar equivalents wrt compound 17) was added drop wise. The reaction wasremoved from the acetone/dry ice bath, warmed to RT, and stirred at RTfor 50 minutes. The reaction was cooled in an acetone/dry ice bath andwater was added drop wise to quench the reaction. The acetone/dry icebath was removed and 20% NaOH (molar equivalents wrt compound 17),dichloromethane, and Rochelle salt (KNa tartrate tetrahydrate) wereadded. The organic layer was separated, the aqueous layer extracted twotimes with dichloromethane, and the organic fractions were combined,washed with water and dried first with K₂CO₃ and then MgSO₄. The driedorganics were filtered and condensed under vacuum to yield clear oil.The clear oil was purified by column chromatography using silica gel and9:1 heptanes: ethyl acetate as the initial eluant then changing to 8:2heptanes: ethyl acetate.

Compound 19: Compound 18 was stirred in dichloromethane, under N₂, andcooled to below 0° C. in an acetone/ice bath. Triethylamine (1.2 molarequivalents wrt compound 18) was added and the reaction cooled to below0° C. Methane sulfonyl chloride (1.3 molar equivalents wrt compound 18)was added slowly while monitoring the temperature to assure that it didnot exceed 5° C. The reaction stirred cold for 1 hour thendichloromethane and water were added. The organic layer was separated,the aqueous layer extracted with dichloromethane, the combined organiclayers were dried with K₂CO₃ and MgSO₄, filtered and condensed undervacuum to yield the mesylate intermediate. The product was used withoutfurther purification.

Compound 20: NaH (1.25 molar equivalents wrt compound 18) was stirredwith dimethyl formamide, under N₂, and a polyamine side chain (1.15molar equivalents wrt compound 18), of chosen length, dissolved intetrahydrofuran was slowly added. The reaction stirred at RT until theevolution of H₂ gas ceased. Starting material mesylate was slowly added(compound 4, step 1 product) and stirred at RT for several hours. Uponcompletion, as evidenced by TLC, the reaction contents were condensedunder vacuum. The crude semi-solid was dissolved in ethyl acetate andwater. The organic layer was separated; the aqueous layer extractedtwice with ethyl acetate, the combined organic layers were washed withwater and condensed under vacuum. The product was purified by columnchromatography using silica gel and 75:25 heptanes: ethyl acetate as theeluant

Compound 21: Compound 20 was stirred in methanol at RT. Concentrated HCl(2 molar equivalents wrt compound 20) was slowly added. The reactionstirred at RT for 30 minutes or until reaction was complete as evidencedby TLC with 60:40 heptanes: ethyl acetate as the mobile phase. Thereaction contents were condensed under vacuum and purified by columnchromatography using silica gel and 95:5 dichloromethane: methanol asthe eluant.

Compound 22: Compound 21 diol was stirred in dichloromethane, under N₂,in an ice/MeOH bath. Benzoyl Chloride (1.03 molar equivalents wrtcompound 21) was added. Once the reaction reached <10° C., pyridine(1.04 molar equivalents wrt compound 21) was slowly added. The reactionwas stirred in the ice/methanol bath for 1 hour and completeness wasdetermined by TLC using 1:1 heptanes: ethyl acetate as the mobile phase.Once reaction was complete, water was added and the reaction stirred for15 minutes in the ice/methanol bath. The organic layer was separated;the aqueous layer extracted with dichloromethane, the combined organiclayers were washed once with water, dried over anhydrous MgSO₄, filteredand condensed under vacuum. The product was purified by columnchromatography using silica gel and 7:3 heptanes: ethyl acetate as theeluant

Compound 23: Compound 22 was stirred in toluene, under N₂, at <5° C.Phosphorus tribromide (1.1 molar equivalents wrt compound 22) was slowlyadded. The reaction was removed from the ice bath and stirred at RT for30 minutes or until the reaction was complete as determined by TLC using95:5 dichloromethane: methanol as the mobile phase. Upon completion thereaction was returned to the ice bath, water was slowly added, and thereaction was stirred for 15 minutes. The organic layer was separated,the aqueous layer extracted two times with ethyl acetate, the combinedorganic layers were washed with 2% (w:v) NaHCO3 and then brine, driedover K₂CO₃ and MgSO₄, filtered and condensed under vacuum. The productwas used without further purification

Compound 24: NaH (1.2 molar equivalents wrt compound 23) was stirred indimethyl formamide, under N₂, at RT and a polyamine side chain (1.2molar equivalents wrt compound 23), of chosen length, dissolved intetrahydrofuran was added slowly. The reaction stirred at RT until theevolution of H₂ gas ceased. Compound 23, dissolved in tetrahydrofuranwas slowly added and the reaction was stirred at RT for several hours.Reaction completeness was determined by TLC using 80:20 toluene: ethylacetate as the mobile phase. The reaction was condensed under vacuum;the crude was dissolved in ethyl acetate and water. The organic layerwas separated, the aqueous layer was extracted with ethyl acetate, andthe combined organic layers were washed with brine, and condensed undervacuum. The product was used without further purification.

Compound 25: Compound 24 was stirred in tetrahydrofuran, under N₂, atRT. Methanol then sodium methoxide (1.5 molar equivalents wrt compound24) were added and the reaction was stirred at RT for 30 minutes.Reaction completeness was determined by TLC using 80:20 toluene: ethylacetate as the mobile phase. Concentrated HCl (molar equivalents wrtsodium methoxide) was added to neutralize the sodium methoxide and thereaction contents were condensed under vacuum. Ethyl acetate and waterwere added to the crude product The organic layer was separated, theaqueous layer washed once with ethyl acetate and once withdichloromethane, the combined organic layers dried with NaSO₄, filteredand condensed under vacuum. The product was purified by columnchromatography using silica gel and 8:2 toluene: ethyl acetate as theeluant.

Scheme 3:

Compound 28: Compound 26 was stirred in dichloromethane, under N₂, at−10° C. in an ice/methanol bath. Triethylamine (2 molar equivalents wrtto compound 26) was added and the reaction was again cooled to −10° C.Methane sulfonyl chloride (2.5 molar equivalents wrt compound 26)dissolved in methylene chloride was added slowly and the reactionstirred cold for 1 hour. Reaction completeness is monitored by TLC using8:2 heptanes: ethyl acetate. Water was slowly added to quench thereaction. The organic layer was separated, the water layer extractedwith dichloromethane, the combined organic layers were washed with brineand condensed under vacuum. The reactive intermediate was usedimmediately without further purification.

Compound 29: Potassium thioacetate (2.5 molar equivalents wrt compound26) in dimethylformamide was stirred, under N₂, at RT. Compound 28mesylate in dimethylformamide was slowly added and the reaction wasstirred overnight The reaction was condensed under vacuum and the solidsdissolved in ethyl acetate and water. The organic layer was separated,the aqueous layer back extracted with ethyl acetate, the combinedorganic layers were washed with brine and condensed under vacuum. Theproduct was purified by column chromatography using silica gel and 8:2toluene: ethyl acetate as the eluant.

Compound 31: NaH (1.25 molar equivalents wrt compound 26) was stirred,under N₂, at RT and dimethylformamide was added. Mesitylene methylsulfonamide dissolved in tetrahydrofuran was slowly added and thereaction was stirred until the evolution of H₂ gas ceased. Compound 28mesylate dissolved in tetrahydrofuran was slowly added and the reactionwas stirred overnight. The reaction was condensed under vacuum and thesolids dissolved in ethyl acetate and water. The organic layer wasseparated, the aqueous layer extracted with ethyl acetate, the combinedorganic layers were washed with brine and condensed under vacuum. Theproduct was purified by column chromatography using silica gel and 8:2toluene: ethyl acetate as the eluant.

Compound 33: NaH (1.25 molar equivalents wrt compound 26) was stirred,under N₂, at RT and dimethylformamide was added. Mesitylene dimethylsulfonamide dissolved in tetrahydrofuran was slowly added and thereaction stirred until the evolution of H₂ gas ceased. Compound 28mesylate dissolved in tetrahydrofuran was slowly added and the reactionwas stirred overnight. The reaction was condensed under vacuum and thesolids dissolved in ethyl acetate and water. The organic layer wasseparated, the aqueous layer extracted with ethyl acetate, the combinedorganic layers were washed with brine and condensed under vacuum. Theproduct was purified by column chromatography using silica gel and 8:2toluene: ethyl acetate as the eluant.

Compound 35: NaH (1.25 molar equivalents wrt compound 26) was stirred,under N₂, at RT and dimethylformamide was added. Mesitylene ethylsulfonamide dissolved in tetrahydrofuran was slowly added and thereaction stirred until the evolution of H2 gas ceased. Compound 28mesylate dissolved in tetrahydrofuran was slowly added and the reactionstirred overnight. The reaction was condensed under vacuum and thesolids dissolved in ethyl acetate and water. The organic layer wasseparated, the aqueous layer extracted with ethyl acetate, the combinedorganic layers were washed with brine and condensed under vacuum. Theproduct was purified by column chromatography using silica gel and 8:2toluene: ethyl acetate as the eluant.

Removal of Mesitylene Protective Groups:

Compounds 30, 32, 34, and 36: Starting material was stirred indichloromethane at RT and phenol (11 molar equivalents per mesitylenegroup) was added. 30% HBr in acetic acid was slowly added (13 molarequivalents per mesitylene group) and the reaction was tightly sealedand stirred for 24-72 hours at RT. Water was added and the reactionstirred for 30 minutes at RT. The organic layer was separated, theaqueous layer was washed five times with dichloromethane, and the waterlayer was condensed under vacuum. 30% NaOH was added to the oil andstirred for several minutes to make the free base. Dichloromethane wasadded and stirred for several more minutes. The organic layer wasseparated, the water layer was extracted five times withdichloromethane, and the combined organic layers were condensed undervacuum. The HCl salt was made by stirring the free base in ethanol andslowly adding concentrated HCl (4 molar equivalents per flee amine). Thereaction was condensed under vacuum and the solids were recrystallizedin a hot ethanol/water mixture.

EXAMPLE 2 Biological Assay for Efficacy In Preventing Alopecia

The efficacy of chemotherapeutic polyamines in reducing or preventingchemotherapy-induced alopecia in a rat model was examined. This animalmodel mimics many of the features found in chemotherapy-induced alopeciaseen in humans and is considered a clinically relevant model for testingnovel therapeutics.

Induction of alopecia by cytoxan (CTX). Lactating Sprague Dawley motherrats with rat pups were purchased from Harlan Sprague Dawley(Indianapolis, Ind.). The mother rats were given food and water adlibitum. The rats pups were tested in the model of chemotherapy-inducedalopecia described by Hussein A. M. et al., Science: 249, 1564 (1990).Cytoxan (CTX), a chemotherapeutic widely used in the treatment ofcancer, was used to induce alopecia in the rats. A common side effect ofcytoxan in patients is alopecia Cytoxan was purchased from SigmaChemicals Co. (St. Louis, Mo.). To produce CTX-induced alopecia, 7 to 10day old rat pups were injected ip. with 35 mg/kg of CTX prepared inphosphate-buffered saline. It was observed that 35 ug/gm of CTX wassufficient to induce 100% hair loss approximately 7 days after cytoxanchallenge.

Chemoprotective polyamines of the invention were prepared in a deliveryvehicle consisting of from 60-100% ethanol in water, depending on thesolubility of the compound. The compounds in ethanol/water solution from50-150 μl in volume were topically administered to the backs of the pupsonce per day before and after CTX challenge. Using a micropipette, theformulation was applied to approximately 2 cm² section of skin to thebacks of the rat pups. Specifically, the pups were treated once dailyfor the 5 days before CTX challenge, once on the day of CTX challengeand once daily for 5 days afterwards. Control groups consisted of pupsreceiving only delivery vehicle. Control groups treated with deliveryvehicle were tested as part of every treatment study. Two or moreanimals were tested per group in both the control and test groups.

Approximately 7 to 10 days after CTX treatment, the pups were evaluatedfor alopecia Hair loss was evaluated using a modified alopecia-scoringindex described by Chen G. et al., Int. J. Cancer: 75, 303 (1998). Ascore of 0=no hair loss; a score of 1=10-30% hair loss; a score of2=40-60% hair loss; a score of 3=70-90% hair loss; and a score of 4=100%hair loss.

EXAMPLE 3 Biological Assay for Efficacy In Preventing Dermatitis

To determine efficacy of chemoprotective polyamines in preventingradiation-induced dermatitis, adult rats were topically treated with thecompounds before and after radiation treatment. Rats were exposed tomedically relevant levels of radiation that could induce clinicalradiation dermatitis. Sprague Dawley rats (Harlen Spraque Dawley) at 4-6weeks-old were anesthetized with sodium pentobarbital at 40 mg/kg bodyweight (Sigma, St Louis, Mo.) prior to radiation exposure. A defined,depilated area on the backs of rats was irradiated using a Mark I, Model30, Cs 137 irradiator (J. L. Sheppard & Associates). Briefly, the backwas stripped of hair to expose the skin using a 1:1 rosin/beeswaxmixture. The rest of the body was protected from radiation exposureusing a lead shield. A dose response study was initially preformed toreproduce relevant dermatitis that matched the Grade (I-IV) scale usedto score the severity of radiation-induced dermatitis in the clinicalsetting. Radiation doses of 5-7 Gray (1 Gray (Gy)=100 mrem) producedGrade I dermatitis within 8-10 days. Radiation doses of 7-10 Gy producedGrade II dermatitis within 8-10 days. After 8-40 days, severe radiationdermatitis was produced at 20-25 Gy (Grade III dermatitis) or at 30-35Gy (Grade IV). Radiation dermatitis of Grade II-III was considered mostclinically relevant, so a radiation challenge dose of 15 Gy in the ratswas used. The stripped back region on the rats was treated topicallywith chemoprotective polyamine once daily for 5 days before and 5 daysafter radiation challenge.

The polyamines were prepared in a delivery vehicle, consisting of from60-100% ethanol in water, depending on the solubility of the compound.The compounds in ethanol/water solution from 100-150 μl in volume weretopically administered to the stripped region. Rats treated with onlythe delivery vehicle served as controls. Eight to ten dayspost-radiation challenge, the rats were evaluated for dermatitis using amodified scoring scale described by Masuda K. et al. Int. J. RadiationOncol. Biol. Phys: 12, 1645(1986). Dermatitis score of 0=normal,1=slight redness, scaly skin with no focal lesions, 2=moderate redness,breakdown of larger area, some small focal lesions, 3=skin very red,breakdown of most of the irradiated area, large ulcers and crustylesions, 4=skin very red, breakdown of the entire irradiated area,severe exudation and large crusty lesions.

EXAMPLE 4 Radiation-Induced Mucositis Model in Hamsters

The model for radiation-induced oral mucositis was developed for thepurpose of screening and identifying effective polyamines useful fortreatment The model used in this example was derived from the oralmucositis model described by Sonis S. T. et al. (Oral Oncology36:373-381, 2000). Male golden Syrian hamsters (70-95 gram, 3542 days,Charles River Laboratories, Wilmington, Mass.) were used. Animals wereindividually numbered, housed in small groups and fed and watered adlibitum. Hamsters were anesthetized with sodium pentobarbital (80 mg/kgbody weight, Sigma, St Louis, Mo.). The left buccal cheek pouch waseverted and secured. A protective lead shield covered the remainder ofthe animal. Subsequently, the cheek pouch was irradiated with a singledose of radiation from 10 to 50 Gy delivered to the targeted mucosa inthe 137 Cs Irradiator. Starting 10 to 12 days after radiation, theseverity of mucositis was assessed every two days. The severity level ofmucositis was evaluated using a modified mucositis scoring systemdescribed by Sonis S. T. et al. (Oral Oncology 36:373-381,2000) Thescoring system was as follows:

-   -   0=Pouch completely healthy. No erythema or vasodilatation.    -   1=Erythema.    -   2=Severe erythema, vasodilatation    -   3=Severe erythema and vasodilatation. Superficial erosion on        radiated pouch surface area.    -   4=Formation of ulcers in one or more places. Cumulative ulcer        formation about up to 50% of radiated pouch surface area        Diminished pliability of mucosa    -   5=Virtually more then 50% or complete ulceration of the radiated        pouch mucosa Loss of pliability.

Manifestations of radiation-induced mucositis were observed by day 12.The hamster buccal pouches were evaluated for the presence of mucositisand photographed every two days from day 12 to day 20. Mucositis wasfound to increase in severity, reaching a peak at day 16. An obviousdose response of radiation was seen, and the grades of mucositis at day16 were scored as: Treatment Mucositis Grade*  0 Gy 0 10 Gy 1 20 Gy 2 30Gy 2.5 40 Gy 4 50 Gy 5*0 = Pouch completely healthy - no erythema or vasodilatation. 1 =Erythema. 2 = Severe erythema, vasodilatation. 3 = Severe erythema andvasodilatation; superficial erosion on radiated pouch surface area. 4 =Formation of ulcers in one or more places; culmulative ulcer formationabout up to 50% of radiated pouch surface area; diminished pliability ofmucosa. 5 = Virtually more than 50% or complete ulceration of theradiated pouch mucosa; loss of pliability.

The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

1. A compound of Formula I:

wherein: each Z is independently A or R¹; each A is independently:

J is a single bond or —CH(Y)—; X is D or —R²-D; Y is H, alkyl, or R³-D;D is —OH, —SH, —SR⁴, or —NR⁴R⁵; each R¹ is independently C₃₋₈ alkylene;each R², R³, R⁶, and R⁷ is independently C₁₄ alkylene; R⁴ is H or loweralkyl; R⁵ is H, lower alkyl, or —R⁶-D; Q is H, lower alkyl, or —R⁷—SR⁴;k is an integer from 2 to about 16; or a stereoisomer, prodrug,pharmaceutically acceptable salt, or mono or polyprotonated acid saltthereof.
 2. A compound of claim 1, wherein each A is independently:


3. A compound of claim 1, wherein each A is independently:


4. A compound of claim 2, wherein Y is H or R³-D.
 5. A compound of claim4, wherein Y is H.
 6. A compound of claim 4, wherein Y is R³-D.
 7. Acompound of claim 5, wherein X is D.
 8. A compound of claim 5, wherein Xis R²-D.
 9. A compound of claim 6, wherein X is D.
 10. A compound ofclaim 6, wherein X is R²-D.
 11. A compound of claim 2, wherein k is aninteger from 2 to
 8. 12. A compound of claim 2, wherein k is an integerfrom 9 to about
 16. 13. A compound of claim 2, wherein k is an integerfrom 9 to
 12. 14. A compound of claim 2, wherein k is
 2. 15. A compoundof claim 2, wherein k is
 3. 16. A compound of claim 2, wherein k is 4.17. A compound of claim 2, wherein k is
 5. 18. A compound of claim 2,wherein k is
 6. 19. A compound of claim 2, wherein k is
 7. 20. Acompound of claim 2, wherein k is
 8. 21. A compound of claim 2, wherein:k is the integer 2; each R¹ is butylene; X is D, D is —NR⁴R⁵, R⁴ is H,and R⁵ is ethyl; and Q is ethyl.
 22. A compound of claim 2, wherein: kis the integer 8; each R¹ is butylene; X is D, D is —NR⁴R⁵, R⁴ is H, andR⁵ is ethyl; and Q is ethyl.
 23. A compound of claim 2, wherein: k isthe integer 2; each R¹ is butylene; X is D, and D is —SH; and Q isethyl.
 24. A compound of claim 2, wherein: k is the integer 4; each R¹is butylene; X is D, and D is —SH; and Q is ethyl.
 25. A compound ofclaim 2, wherein: k is the integer 6; each R1 is butylene; X is D, and Dis —SH; and Q is ethyl.
 26. A compound of claim 2, wherein: k is theinteger 8; each R1 is butylene; X is D, and D is —SH; and Q is ethyl.27. A compound of claim 2, wherein: k is the integer 4; each R¹ isbutylene; X is D, D is —NR⁴R⁵, R⁴ is H, and R⁵ is methyl; and Q isethyl.
 28. A compound of claim 4 wherein Q is H or lower alkyl.
 29. Acompound of claim 3, wherein Y is H or R³-D.
 30. A compound of claim 29,wherein Y is H.
 31. A compound of claim 29, wherein Y is R³-D.
 32. Acompound of claim 30, wherein X is D.
 33. A compound of claim 30,wherein X is R²-D.
 34. A compound of claim 31, wherein X is D.
 35. Acompound of claim 31, wherein X is R²-D.
 36. A compound of claim 3,wherein k is an integer from 2 to
 8. 37. A compound of claim 3, whereink is an integer from 9 to about
 16. 38. A compound of claim 3, wherein kis an integer from 9 to
 12. 39. A compound of claim 3, wherein k is 2.40. A compound of claim 3, wherein k is
 3. 41. A compound of claim 3,wherein k is
 4. 42. A compound of claim 3, wherein k is
 5. 43. Acompound of claim 3, wherein k is
 6. 44. A compound of claim 3, whereink is
 7. 45. A compound of claim 3, wherein k is
 8. 46. A compound ofclaim 29 wherein Q is H or lower alky.
 47. A compound of claim 3 whereinJ is a single bond.
 48. A compound of claim 3 wherein J is —CH(Y)—. 49.A pharmaceutical preparation for reducing or preventing hair loss,dermatitis, mucositis or gastrointestinal distress caused by treatmentwith a chemotherapeutic agent or radiation therapy, which comprises atleast one compound of Formula I and a topical delivery vehicle forlocally delivering the compound to dermal or mucosal cells of skin,scalp, mouth, nasoesophageal, gastrointestinal or urogenital system,wherein Formula I is

wherein: each Z is independently A or R1; each A is independently:

J is a single bond or —CH(Y)—; X is D or —R²-D; Y is H, alkyl, or R³-D;D is —OH, —SH, —SR⁴, or —NR⁴R; each R¹ is independently C₃₋₈ alkylene;each R², R³, R⁶, and R⁷ is independently C₁₋₆ alkylene; R⁴ is H or loweralkyl; R⁵ is H, lower alkyl, or —R⁶-D; Q is H, lower alkyl, or —R⁷—SR⁴;k is an integer from 2 to about 16; or a stereoisomer, prodrug,pharmaceutically acceptable salt, or mono or polyprotonated acid saltthereof.
 50. The pharmaceutical preparation of claim 49, which fathercomprises at least one other agent that reduces or prevents hair loss,dermatitis, mucositis or gastrointestinal distress caused by treatmentwith a chemotherapeutic agent or radiation therapy.
 51. Thepharmaceutical preparation of claim 50, wherein the other agent is ananti-proliferative agent.
 52. The pharmaceutical preparation of claim50, wherein the other agent is a chemoprotective inducing agent.
 53. Thepharmaceutical preparation of claim 50, wherein the other agent is afree radical scavenger.
 54. The pharmaceutical preparation of claim 49,wherein the topical delivery vehicle comprises one or more of liposomes,lipid droplet emulsions, oils, aqueous emulsions of polyoxyethyleneethers, aqueous alcohol mixtures, aqueous ethanol mixtures containingpropylene glycol, aqueous ethanol mixtures containing phosphatidylcholine, lysophosphatidyl choline and triglycerides, xanthan gum inaqueous buffer, hydroxypropymethylcellulose in aqueous buffer or aqueousalcohol mixtures, diethylene glycol monoethyl ether in aqueous buffer,and biodegradable microparticles.
 55. The pharmaceutical preparation ofclaim 54, formulated for topical delivery to skin or hair follicles,wherein the delivery vehicle comprises an aqueous alcohol mixture. 56.The pharmaceutical preparation of claim 55, wherein the delivery vehiclefurther comprises propylene glycol.
 57. The pharmaceutical preparationof claim 56, formulated as a cream, lotion, ointment or gel.
 58. Thepharmaceutical preparation of claim 54, formulated for topical deliveryto the oral cavity or naso-esophageal passages, wherein the deliveryvehicle comprises a mucoadhesive substance.
 59. The pharmaceuticalpreparation of claim 58, formulated as an aerosol, oral rinse, ointmentor gel.
 60. The pharmaceutical preparation of claim 54, formulated forvaginal or rectal delivery, wherein the delivery vehicle comprises amucoadhesive substance.
 61. The pharmaceutical preparation of claim 60,formulated as a cream, ointment, lotion, gel, foam or suppository. 62.The pharmaceutical preparation of claim 54, formulated for topicaldelivery to the gastrointestinal tract, wherein the delivery vehiclecomprises one or more of nonionic liposomes and mucoadhesive substances.63. The pharmaceutical preparation of claim 62, formulated as a liquidfor coating the surface of the gastrointestinal tract.
 64. A method forreducing or preventing hair loss dermatitis, mucositis orgastrointestinal distress in a patient undergoing treatment with achemotherapeutic agent or radiation therapy, which comprisesadministering to the patient a prophylactically or therapeuticallyeffective amount of a pharmaceutical preparation comprising at least onecompound of Formula I and a topical delivery vehicle for locallydelivering the compound to dermal or mucosal cells of skin, scalp,mouth, nasoesophageal, gastrointestinal or urogenital system, whereinFormula I is:

wherein: each Z is independently A or R¹; each A is independently:

J is a single bond or —CH(Y)—; X is D or —R²-D; Y is H, alkyl, or R³-D;D is —OH, —SH, —SR⁴, or —NR⁴R⁵; each R¹ is independently C₃₋₈ alkylene;each R², R³, R⁶, and R⁷ is independently C₁₋₆ alkylene; R⁴ is H or loweralkyl; R¹ is H, lower alkyl, or —R⁶-D; Q is H, lower alkyl, or —R⁷—SR⁴;k is an integer from 2 to about 16; or a stereoisomer, prodrug,pharmaceutically acceptable salt, or mono or polyprotonated acid saltthereof.
 65. The method of claim 64, wherein the pharmaceuticalpreparation is administered beginning at least one day prior tochemotherapy or radiation therapy.
 66. The method of claim 65, whereinthe pharmaceutical preparation is administered beginning at least fivedays prior to chemotherapy or radiation therapy.
 67. The method of claim64, wherein the pharmaceutical preparation is administered afterinitiation of chemotherapy or radiation therapy.
 68. The method of claim64, wherein the pharmaceutical preparation is administered throughout acourse of chemotherapy or radiation therapy.
 69. The method of claim 64,wherein the pharmaceutical preparation is administered followingtermination of a course of chemotherapy or radiation therapy.
 70. Themethod of claim 64, which further comprises administering to the patientat least one other agent that reduces or prevents hair loss, dermatitis,mucositis or gastrointestinal distress caused by treatment with achemotherapeutic agent or radiation therapy.
 71. The pharmaceuticalpreparation of claim 70, wherein the other agent is ananti-proliferative agent.
 72. The pharmaceutical preparation of claim70, wherein the other agent is a chemoprotective inducing agent.
 73. Thepharmaceutical preparation of claim 70, wherein the other agent is afree radical scavenger.
 74. A method of treating cancer that increases apatient's tolerance to high doses of a chemotherapeutic agent orradiation therapy, the method comprising: a) administering the high doseof the chemotherapeutic agent or radiation therapy to the patient; andb) administering one or more pharmaceutical preparations for reducing orpreventing one or more of chemotherapy- or radiation therapy-inducedhair loss, dermatitis, mucositis or gastrointestinal distress, in anamount and for a time to reduce or prevent the one or more of thechemotherapy- or radiation therapy-induced hair loss, dermatitis,mucositis or gastrointestinal distress, thereby increasing the patient'stolerance to the high dose of the chemotherapeutic agent or radiationtherapy, wherein the pharmaceutical preparation comprises compound ofFormula I and a topical delivery vehicle for locally delivering thecompound to dermal or mucosal cells of skin, scalp, mouth,nasoesophageal, gastrointestinal or urogenital system, wherein Formula Iis:

wherein: each Z is independently A or R¹; each A is independently:

J is a single bond or —CH(Y)—; X is D or —R²-D; Y is H, alkyl, or R³-D;D is —OH, —SH, —SR⁴, or —NR⁴R⁵; each R¹ is independently C₃₋₈ alkylene;each R², R³, R⁶, and R⁷ is independently C-6 alkylene; R⁴ is H or loweralkyl; R⁵ is H, lower alkyl, or —R⁶-D; Q is H, lower alkyl, or —R⁷—SR⁴;k is an integer from 2 to about 16; or a stereoisomer, prodrug,pharmaceutically acceptable salt, or mono or polyprotonated acid saltthereof.